Utrophin gene expression

ABSTRACT

Nucleic acid from which a polypeptide with utrophin function can be expressed, especially mini-genes and chimaeric constructs. Expression significantly decreases the severity of the dystrophic muscle phenotype in an animal model, indicating usefulness in treatment of muscular dystrophy. The nucleic acid and encoded polypeptides are also useful in screening for substances to modulate utrophin binding to actin and/or the dystrophin protein complex.

The present invention generally relates to the provision of nucleic acid from which a polypeptide with utrophin function can be expressed, especially mini-genes and chimaeric constructs. Expression of a utrophin transgene significantly decreases the severity of the dystrophic muscle phenotype in an animal model.

The severe muscle wasting disorders, Duchenne muscular dystrophy (DMD) and the less debilitating Becker muscular dystrophy (BMD) are due to mutations in the dystrophin gene. Dystrophin is a large cytoskeletal protein which in muscle is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). The protein is composed of four domains: an actin-binding domain (shown in vitro to bind actin), a rod domain containing triple helical repeats, a cysteine rich (CR) domain and a carboxy-terminal (CT) domain. The majority of the CRCT binds to a complex of proteins and glycoproteins (called the dystrophin protein complex, DPC) spanning the sarcolemma. This complex consists of cytoskeletal syntrophins and dystrobrevin, transmembrane, β-dystroglycan, α-, β-δ-, γ-sarcoglycans and extracellular α-dystroglycan. The DPC links to laminin-α2 (merosin) in the extracellular matrix and to the actin cytoskeleton via dystrophin within the cell. The breakdown of the integrity of the DPC due to the loss of, or impairment of dystrophin function, leads to muscle degeneration and the DMD phenotype. The structure of dystrophin and protein interactions within the DPC have been recently reviewed [1,2,3].

There are various approaches which can be adopted for the gene therapy of DMD. These include myoblast transfer, retroviral infection, adenoviral infection and direct injection of plasmid DNA. In most cases the dystrophin gene used in the experiments generates a truncated protein approximately half the size of the full size protein. This dystrophin minigene was modelled on a natural mutation identified in a very mild Becker patient [4]. The cloned version of this truncated minigene is able to reverse the pathological phenotype in the dystrophin deficient mdx mouse [5,6,7] and has had limited success when delivered to mdx muscle by viral vectors [8,9,10]. Although some progress is being made in each of these areas using the mdx mouse as a model system, there are problems related to the number of muscle cells that can be made dystrophin positive, the levels of expression of the gene and the duration of expression [11]. Another problem to be addressed is the rejection of cells expressing dystrophin because of immunological intolerance i.e. dystrophin within these cells will appear foreign to the host immune system given that most DMD patients will never have expressed dystrophin [12,13].

In order to circumvent some of these problems, possibilities of compensating for dystrophin loss using a related protein, utrophin, are being explored.

Utrophin is a 395 kDa protein encoded by a gene located on chromosome 6q24 and shown to have strong sequence similarity to dystrophin [14]. The actin binding domain of dystrophin and utrophin has 85% similarity and the DPC binding region has 88% similarity. Both of these domains have been shown to function as predicted in vitro. The structure and potential protein interactions are described in detail in reviews [1,2,3].

There is a substantial body of evidence demonstrating that utrophin is capable of localising to the sarcolemma. During normal fetal muscle development there is increased utrophin expression, localised to the sarcolemma up until 18 weeks and 20 days gestation in human and mouse respectively. After this time the utrophin sarcolemmal staining steadily decreases to the significantly lower adult levels shortly before birth where utrophin is localised almost exclusively to the NMJ and MTJ [15,16,17]. The decrease in utrophin expression coincides with increased expression of dystrophin [17]. Many studies have shown that utrophin is bound to the sarcolemma in DMD and BMD patients. However the levels of utrophin localised at the sarcolemma vary from report to report [18,19,20,21]. In some other non Xp21 myopathies, utrophin and dystrophin are simultaneously bound to the sarcolemma of adult skeletal muscle [22].

High levels of utrophin may protect muscle from the consequences of dystrophin loss. Matsumara et al. [23] demonstrated that purified membranes from the mdx mouse contained complexes of utrophin and the DPC. When quadricep muscles (which show necrosis) from these mice were analysed by immunoblotting, the level of utrophin remained approximately the same, however the level of the α-dystroglycan was drastically reduced. In cardiac muscle (which shows no pathology) the level of utrophin was elevated four fold with no loss of the α-dystroglycan. Immunocytochemical analysis of other mdx small calibre skeletal muscles (extraocular and toe) which also have no pathology shows increased utrophin expression and normal levels of α-sarcoglycan. This result suggests that the increased levels of utrophin interacts with the DPC (or an antigenically related complex) at the sarcolemma and prevents loss of the complex thus the structure of these cells remains normal. In the mdx mouse, utrophin levels in muscle remain elevated soon after birth compared with normal mice; however once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected [15,16].

Thus, in certain circumstances utrophin can localise to the sarcolemma probably at the same binding sites as dystrophin, namely actin and the DPC. If the expression of utrophin is high enough, it may maintain the DPC and thus alleviate the DMD phenotype. It is unlikely that such external upregulation could be tightly controlled giving rise to potentially high levels of utrophin within the cell. However, this may not be a problem as Cox et al. [24] have demonstrated that gross over expression of dystrophin in the muscle of transgenic mdx mice reverts the muscle pathology to normal with no obvious detrimental side effects.

The present invention has arisen from cloning of nucleic acid encoding utrophin and fragments of utrophin from various species. The original aim was to clone nucleic acid encoding human utrophin, but major problems were encountered. A previous paper (14) reported the amino acid sequence of utrophin (so-called “dystrophin-related protein”), obtained by cloning of overlapping cDNAs. However, two regions around the amino terminal actin binding domain were not represented in these clones. These regions could be amplified by PCR and sequenced, but it has proved not to be possible to clone them. Either clones which should have included these regions were rearranged (as determined by restriction mapping) or simply no clones were isolated even if highly recombination deficient E. coli host strains (SURE and STBL2) were used. The gaps in the sequence were identified by comparing the sequence generated from the utrophin cDNAs to the published human dystrophin sequence. It became apparent as further utrophin clones were isolated, none spanned these two gaps.

Sequence information obtained from the amino terminus of the human cDNA was used to design probes and rat and mouse cDNA libraries were screened. Rat cDNAs were also unstable or rearranged in the region corresponding to the unclonable regions in the human sequence. Some large rat clones covering these regions were obtained, but all attempts to generate subclones failed due to rearrangements of the inserts as determined by restriction mapping. Surprisingly, in view of the difficulties with the human and rat sequences, cDNA from the mouse library, covering the regions in question, was found to be stable and amenable to further manipulation including the. generation of smaller subclones.

FIG. 1 shows a comparison between human, rat and mouse utrophin nucleotide sequences encoding part of the amino-terminal portion of the respective proteins. The unclonable regions of the human gene are underlined.

This cloning work enables for the first time the construction of a nucleic acid molecule from which a polypeptide with utrophin function can be expressed.

Furthermore, by way of analogy with the success achieved with a dystrophin mini-gene (from which a truncated version of dystrophin is expressed) the present invention provides “utrophin mini-genes” and polypeptides encoded thereby. To overcome the problem of unclonability of regions of the human utrophin gene sequence, the present inventors have realised that it is possible to employ a sequence of nucleotides derived from the mouse utrophin gene in a chimaeric construct to provide for expression of a polypeptide with utrophin function.

According to a first aspect of the present invention there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding a polypeptide with utrophin function.

A potypetide with utrophin function is able to bind actin and able to bind the dystrophin protein complex (DPC).

Polypeptides with utrophin function are generally distinguishable immunologically from dystrophin polypeptides. For example, they may comprise at least one epitope not found in dystrophin. Polypeptides with utrophin function may be identified using specific polyclonal or monoclonal antibodies which do not cross-react with dystrophin. If a polypeptide is able to bind actin and able to bind the dystrophin protein complex and at least one antibody can bind it which cannot bind dystrophin, then the polypeptide has utrophin function. In a preferred embodiment, the polypeptide can be bound by an antibody which binds utrophin but not dystrophin, in other words the polypeptide shares at least one epitope with utrophin which epitope is not found in dystrophin. In another embodiment, the polypeptide does not contain an epitope found in dystrophin, such that the polypeptide is not bound by an antibody which binds dystrophin. In such a case, the epitope recognised by the antibody which binds dystrophin may be one not found in utrophin. The polypeptide may contain no epitope found in dystrophin. The immunological comparison may be made with human utrophin and/or dystrophin, especially if the polypeptide with utrophin function is intended for human use, or with the utrophin and/or dystrophin of the species in which use is intended, e.g. mouse. Mouse monoclonal antibodies MANCH07 and MANNUT1 [31] were used in the work described herein. Standard in vitro binding assays may be used to assess immunological cross-reactivity of a polypeptide.

Thus, the polypeptide comprises an actin-binding domain and a dystrophin protein complex (DPC)-binding domain and utrophin-like as opposed to dystrophin-like, e.g. as determined immunologically.

Preferably the encoding sequence comprises a human sequence, i.e. a sequence obtainable from the genome of a human cell.

Comparison of various amino acid sequences reveals the following % similarities (calculated using the method of Needleman and Wunsch (1974) J. Mol. Biol. 48: 443-453, performed using the GAP program from the Winsconsin Package v8, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711, USA) and identities:

full length human dystrophin v. human utrophin 69% similarity, 50.7% identity;

full length human utrophin v. rat utrophin 93.2% similarity, 87.1% identity;

full length human dystrophin v. mouse dystrophin 95.4% similarity, 91.2% identity;

human dystrophin C-terminus v. human utrophin C-terminus 84.1% similarity, 73.6% identity.

As noted, the present invention is only concerned with “utrophin-like” molecules, not “dystrophin-like” molecules. Thus, polypeptides according to the present invention (e.g. as encoded by nucleic acid according to the invention) may have an amino acid sequence which is greater than about 75% similar, preferably greater than about 80%, about 85%, about 90%, about 95% or about 98% similarity to the amino acid sequence of FIG. 3 or the amino acid sequence of FIG. 9, taken over the full length. The polypeptides may have an amino acid identity of greater than about 55% identity, preferably greater than about 60% identity, about 70%, about 80%, about 90%, about 95% or about 98% identity over the full-length. The levels of similarity and/or identity may be lower outside the C-terminal, DPC-binding domain provided the DPC-binding domain has greater than about 85% similarity, preferably greater than about 90%, about 95% or about 98% similarity with the DPC-binding domain amino acid sequence of FIG. 3 or FIG. 9, or has greater than about 80%, preferably greater than about 85%, about 90%, about 95% or about 98% identity with the DPC-binding domain amino acid sequence of FIG. 3 or FIG. 9. Particular amino acid sequence variants or derivatives may have a sequence which differs from the sequence of FIG. 3 or FIG. 9 by one or more of insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-50, 50-100, 100-150, or more than 150 amino acids.

The nucleic acid molecule may be an isolate, or in an isolated and/or purified form, that is to say not in an environment in which it is found in nature, removed from its natural environment. It may be free from other nucleic acid obtainable from the same species, e.g. encoding another polypeptide.

The nucleic acid molecule may be one which is not found in nature. For example, the sequence of nucleotides may form part of a cloning vector and/or an expression vector, as discussed further below. The sequence of nucleotides may represent a variant or derivative of a naturally occurring sequence by virtue of comprising an addition, insertion, deletion and/or substitution of one or more nucleotides with respect to the natural sequence, provided preferably that the encoded polypeptide has the specified characteristics. The addition, insertion, deletion and/or substitution of one or more nucleotides may or may not be reflected in an alteration in the encoded amino acid sequence, depending on the genetic code.

Preferably, the nucleic acid molecule is a “mini-gene”, i.e. the polypeptide encoded does not correspond to full-length utrophin but is rather shorter, a truncated version. For instance, part or all of the rod domain may be missing, such that the polypeptide comprises an actin-binding domain and a DPC-binding domain but is shorter than naturally occurring utrophin. In a full-length utrophin gene, the actin-binding domain is encoded by nucleotides 1-739, while the DPC-binding domain (CRCT) is encoded by nucleotides 8499-10301 (where 1 represents the start of translation; FIG. 2A). The respective domains in the polypeptide encoded by a mini-gene according to the invention may comprise amino acids corresponding to those encoded by these nucleotides in the full-length coding sequence.

Dystrophin mini-genes have been shown to be active in animal models (as discussed). Advantages of a mini-gene over a sequence encoding a full-length utrophin molecule or derivative thereof include easier manipulation and inclusion in vectors, such as adenoviral and retroviral vectors for delivery and expression.

A further preferred non-naturally occurring molecule encoding a polypeptide with the specified characteristics is a chimaeric construct wherein the encoding sequence comprises a sequence obtainable from one mammal, preferably human (“a human sequence”), and a sequence obtainable from another mammal, preferably mouse (“a mouse sequence”). Such a chimaeric construct may of course comprise the addition, insertion, substitution and/or deletion of one or more nucleotides with respect to the parent mammalian sequences from which it is derived. Preferably, the part of the coding sequence which encodes the actin-binding domain comprises a sequence of nucleotides obtainable from the mouse, or other non-human mammal, or a sequence of nucleotides derived from a sequence obtainable from the mouse, or other non-human mammal.

In a preferred embodiment, the sequence of nucleotides encoding the polypeptide comprises sequence GAGGCAC at residues 332-338 and/or the sequence GATTGTGGATGAAAACAGTGGG at residues 1452-1476 (using the conventional numbering from the initiation codon ATG), and a sequence obtainable from a human.

The nucleic acid molecule may comprise a nucleotide sequence encoding a sequence of amino acids shown in FIG. 1. As discussed, the encoding sequence may be chimaeric, i.e. comprise sequences of nucleotides from different species, e.g. a sequence from or derivable from a human and a sequence from or derivable from a mouse or other non-human mammal.

A chimaeric mini-gene encoding sequence according to the present invention is shown in FIG. 3. Preferred embodiments of the present invention include a nucleic acid molecule comprising a sequence of nucleotides encoding a polypeptide which has an actin-binding domain and a DPC-binding domain and which polypeptide comprises an amino acid sequence encoded by a sequence of nucleotides shown in FIG. 3, a nucleic acid molecule comprising a sequence of nucleotides encoding a variant, allele. or derivative of such a polypeptide by way of addition, substitution, insertion and/or deletion of one or more amino acids, and a nucleic acid molecule comprising a sequence of nucleotides which is a variant, allele or derivative of the sequence shown in FIG. 3, by way of addition, substitution, insertion and/or deletion of one or more nucleotides, with or without a change in the encoded amino acid sequence with respect to the amino acid sequence encoded by a sequence of nucleotides shown in FIG. 3. The proviso is that the encoded polypeptide is “utrophin-like” rather than “dystrophin-like”, e.g. as determined immunologically as discussed.

One particular variant or derivative of the sequence of FIG. 3 has a sequence as shown in FIG. 9, which is a “full-length” utrophin construct, including rod domain sequences not included in the mini-gene of FIG. 3.

The sequences of FIG. 3 and FIG. 9 include some positions at which the precise residue is left open (marked by “N” in the nucleotide sequence and “X” in the amino acid sequence). Comparison of the human, mouse and rat utrophin sequences in this region (FIG. 10) shows that the human and rat amino acid sequences are absolutely conserved here. Accordingly, the twelve “X's” in FIGS. 3 and 9 may represent the amino acid sequence DKKSIIMYLTSL (SEQ ID NO:15). Instead, in accordance with the discussion of variants and derivatives herein, a polypeptide according to the invention (as encoded by nucleic acid according to the invention) may include a variant or derivative sequence, by way of one or more of insertion, addition, substitution or deletion of one or more amino acids of the sequence DKKSIIMYLTSL (SEQ ID NO:15), in the position marked by the X's in FIGS. 3 and 9.

Nucleic acid according to the present invention is obtainable by hybridising nucleic acid of target cells (e.g. human, mouse, rat) with one or more oligo- or poly-nucleotides with sequences designed based on the sequence information presented in FIG. 1, FIG. 3 or FIG. 9. Thus, the full mouse sequence, or the sequence in the region marked by the X's in FIGS. 3 and 9, may be obtained by probing or PCR using sequence information provided herein (e.g. FIG. 1).

Nucleic acid according to the present invention is obtainable using one or more oligonucleotide probes or primers designed to hybridise with one or more fragments of a nucleic acid sequence shown in FIG. 1, FIG. 3 or FIG. 9, particularly fragments of relatively rare sequence, based on codon usage or statistical analysis. The amino acid sequence information provided may be used in design of degenerate probes/primers or “long” probes. A primer designed to hybridise with a fragment of the nucleic acid sequence shown may be used in conjunction with one or more oligonucleotides designed to hybridise to a sequence in a cloning vector within which target nucleic acid has been cloned, or in so-called “RACE” (rapid amplification of cDNA ends) in which cDNA's in a library are ligated to an oligonucleotide linker and PCR is performed using a primer which hybridises with the sequence shown in the figure and a primer which hybridises to the oligonucleotide linker.

Nucleic acid isolated and/or purified from one or more cells (e.g. human, mouse) or a nucleic acid library derived from nucleic acid isolated and/or purified from cells (e.g. a cDNA library derived from mRNA isolated from the cells), may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR).

A method may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.

Preliminary experiments may be performed by hybridising under low stringency conditions various probes to Southern blots of DNA digested with restriction enzymes. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched.

It may be necessary for one or more gene fragments to be ligated to generate a full-length coding sequence. Also, where a full-length encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries.

Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. A common, preferred bacterial host is E. coli.

Nucleic acid according to the present invention may form part of a cloning vector and/or a vector from which the encoded polypeptide may be expressed. Suitable vectors can be chosen or constructed, containing appropriate and appropriately positioned regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2 nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

The invention also provides a mammal, such as a human, primate or rodent, preferably rat or mouse, comprising a host cell as provided, and methods of production and use of such a mammal. The mammal may be non-human. Transgenic animals, particularly mice, can be generated using any available technique. Particularly suitable for purposes of study are mdx mice or others with a dystrophic phenotype.

The polypeptide encoded by the nucleic acid may be expressed from the nucleic acid in vitro, e.g. in a cell-free system or in cultured cells, or in vivo. In vitro expression may be useful in determining ability of the polypeptide to bind to actin and/or DPC. This may be useful in testing or screening for substances able to modulate one or both of these binding activities. In particular, substances able to increase actin and/or DPC binding of the polypeptide will add to the repertoire of molecules available for potential pharmaceutical/therapeutic exploitation. Such substances, identified as modulators of one or both of the binding activities of the polypeptide, following expression of the polypeptide from encoding nucleic acid therefor, may be investigated further and may be manufactured and/or used in preparation of a medicament, pharmaceutical composition or drug which may subsequently be administered to an individual. In vivo expression is discussed further below.

According to a further aspect of the present invention there is provided a polypeptide with utrophin function (other than utrophin itself). Such a polypeptide comprises an actin-binding domain and a DPC-binding domain and is immunologically recognisable as utrophin-like rather than dystrophin-like, as discussed, not-being a naturally occurring polypeptide. The polypeptide may be any of those discussed above as being encoded by nucleic acid according to the present invention. In particular, the polypeptide may be shorter than naturally occurring full-length utrophin, for example by virtue of lacking all or part of the rod domain. The actin-binding and DPC-binding domains may correspond to those of human, mouse or other non-human utrophin or may be derived therefrom by way of addition, substitution, insertion and/or deletion of one or more amino acids. The polypeptide may be chimaeric, comprising sequences of amino acids from or derived from different species, e.g. human and mouse, as discussed.

A convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it. Accordingly, methods of making such polypeptides by expression from encoding nucleic acid therefor are provided by the present invention, in vitro, e.g. in cell-free systems or by culturing host cells under appropriate conditions for expression, or in vivo.

Polypeptides and nucleic acid according to the invention may be used in the manufacture of medicaments, compositions, including pharmaceutical formulations, and drugs for delivery to an-individual, e.g. a human with muscular dystrophy or a non-human mammal, such as a mouse, as a model for study of the polypeptides, muscular dystrophy and therapy thereof.

For example, a method of treatment practised on the human or animal body in accordance with the present invention may comprise administration to an individual of nucleic acid encoding a polypeptide as disclosed herein. The nucleic acid may form part of a construct enabling expression within cells of the individual. Nucleic acid may be introduced into cells using a retroviral vector, preferably one which will not transform cells, or using liposome technology.

Administration is preferably in a “therapeutically effective amount”, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such has water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Injection may be used to deliver nucleic acid to disease sites. Internally, suitable imaging devices may be employed to guide an injecting needle to the desired site.

It may be desirable to remove cells from the body, treat them then return them to the body, or to administer cells derived from cells removed from an individual. This might be appropriate, for example, if muscle stem cells can be isolated. Muscle precursor cells (“mpc”) have been used in cell therapy in mdx mice, where implantation of normal mpc gave rise to substantial amounts of dystrophin [25,26,27]. Immunosuppression increases success of cell implantation procedures [13]. Myoblasts may be used to introduce genes into muscle fibres during growth or repair, as has been demonstrated using a replication-defective retroviral vector to introduce a mini-dystrophin construct into proliferating myogenic cells in tissue culture [28].

Thus, cells in culture may have nucleic acid according to the present invention introduced into them before the cells are grafted into muscles in a patient. Grafting the cells back into the donor has the advantages of a genetically corrected autologous transplant. Nucleic acid may be introduced locally into cells using transfection, electroporation, microinjection, liposomes, lipofecting or as naked DNA or RNA, or using any other suitable technique.

Retroviral vectors have also been used to introduce the dystrophin mini-gene into the myoblasts of spontaneously regenerating muscle of the mdx mouse to produce dystrophin-positive fibres [8]. Recombinant replication defective adenoviruses appear particularly effective as an efficient means of introducing constructs into skeletal muscle fibres for persistent expression [29]. See reference 11 for a review of myoblast-based gene therapies.

Adenoviral, retroviral or other viral vectors may be used advantageously for the introduction of a utrophin sequence according to the present invention into muscle cells. Even though in vivo transduction may be restricted to growing or regenerating muscle fibres, retrovirally introduced constructs have the advantage of becoming integrated into the genome of the host cell, potentially conferring lifelong expression.

Liposomes may be used as vehicles for delivery of nucleic acid constructs to skeletal muscle. Intravenous injection of constructs in cationic liposomes has resulted in widespread transfection of most tissues, including skeletal muscle [30]. Lack of immunogenicity allows for repeated administration and lack of tissue specificity may be accommodated by choosing a muscle-specific promoter to drive expression.

For use in distinguishing polypeptide with utrophin function from dystrophin and related polypeptides, antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (eg mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof, or a cell or virus which expresses the protein or fragment. Immunisation with DNA encoding a target polypeptide is also possible (see for example Wolff, et al. Science 247: 1465-1468 (1990); Tang, et al. Nature 356: 152-154 (1992); Ulmer J B, et al. Science 259: 1745-1749 (1993)). Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82).

The production of monoclonal antibodies is well established in the art. Monoclonal antibodies can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. A hybridoma producing a monoclonal antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, eg using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces;. for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the target, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest (or a fragment thereof).

Antibodies may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any specific binding substance having an binding domain with the required specificity. Thus this covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.

It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

Further aspects and embodiments of the present invention, and modifications to aspects and embodiments disclosed herein, will be apparent to those skilled in the art.

The following figures are attached hereto:

FIGS. 1a and 1 b: FIG 1 a shows the corresponding parts of nucleotide sequences of the mouse (Moutro) (SEQ ID NO:1), rat (ratutro) (SEQ ID NO:2) and human (humutro) (SEQ ID NO:3) starting from the first amino acid and encompassing the actin binding domain and start of the rod domain. The heavyline represents the unclonable region in rat and human. FIG. 1b shows the sequence of the second unclonable region (SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6).

FIGS. 2a-2 c are a schematic of the cloning process of PCR6.0. FIG. 2a: The bold numbers represent the utrophin transcript in kb and the numbers below the line represent the nucleotide positions of the regions in question where 1 is the start of translation; FIG. 2b represents the cDNAs used as template for the PCR; FIG. 2c represents the two PCR fragments generated to form PCR6.0.

FIGS. 3a-3 e show the nucleotide sequence (both strands) (SEQ ID NO:7 and SEQ ID NO:8) of a “utrophin mini-gene” according to the present invention and whose construction is described herein.

FIG. 4 shows a representation of the dystrophin and utrophin polypeptides, showing the various domains, and “mini-genes” comprising only parts of the full-length molecules.

FIGS. 5a-5 c: Utrophin transgene construction and expression. FIG. 5a, Scale representation of dystrophin, utrophin and the two truncated transgenes. The repeated spectrin-like repeats (R) and the potential hinge sites (H) are marked. FIG. 5b, Utrophin transgene vector. The N- and C-terminal portions of utrophin were cloned as PCR products using overlapping cDNAs as template. The regions used are indicated by the dotted lines. The PCR product was cloned into a vector containing the 2.2 kb human skeletal α-actin (HSA) promoter and regulatory regions ^(20,21) and SV40 large T poly A site. The cloning sites were such that the transgene was located near the beginning of the second HSA untranslated exon and the Asp718/NotI sites were used to liberate the complete fragment. FIG. 5c, Immunoblot of muscle from the utrophin transgenic line F-3 and a non-transgenic C57BL/10 littermate. M, skeletal muscle; H, heart; D, diaphragm.

FIGS. 6a and 6 b: Decrease in serum CK levels and centralised myofibres in transgenic mdx mice. FIG. 6a, Serum creatine kinase levels in male mdx mice expressing the utrophin transgene. Serum creatine kinase levels from 5 week old mice generated from 4 F₃ litters resultant from a male transgenic mouse crossed with female mdx. Offspring consisted of male hemizygous mdx (M mdx), male utrophin transgenic mdx (M Tg mdx) and heterozygous females (F mdx). Female heterozygotes are not significantly different from wild type so can be used as normal controls. The number of mice (n) in each group is shown in parentheses and the mean SE shown by T-bars. FIG. 6b, Proportion of myofibres containing centralised nuclei. The mean SE is shown by T-bars.

FIGS. 7a and 7 b: Decrease in centralised nuclei in Diaphragm and TA muscle from other truncated utrophin transgenic mdx lines (Gerald, George, Grant, Gavin), normal (n) and mdx (mdx). The mean SE is shown by T-bars.

FIG. 8: Decrease in serum creatine kinase from other utrophin transgenic lines, normal (n) and mdx (mdx). The number of mice in each group is shown in parenthesis.

FIGS. 9a-9 i: Full length utrophin coding sequence (SEQ ID NO:9) and encoded amino acid sequence (SEQ ID NO:10).

FIG. 10: Alignment of amino acid sequences for the N-terminal regions of human (SEQ ID NO:11), mouse (SEQ ID NO:12) and rat (SEQ ID NO:13) utrophin.

All documents cited are incorporated herein by reference.

EXAMPLE 1 Cloning of Utrophin Minigenes

The four cDNAs covering the latter half of the human utrophin transcript were ligated together using overlapping restriction endonuclease sites. The amino-terminal region was reconstructed using the human 92.2 cDNA joined by the common EcoRI restriction site to the stable mouse cDNA clone, JT1. These two constructs were then used as templates for PCR amplification (FIG. 2B). Primers were designed to generate two fragments, PCR2.0 and PCR 4.0, containing no untranslated regions which could be ligated in frame to generate a utrophin minigene containing approximately the first 2 kb and last 4 kb of the utrophin coding sequence (FIG. 2C).

The two PCR fragments were ligated together using the HpaI site. The complete DNA sequence of the 6.0 kb minigene is shown in FIG. 3. The complete 6 kb minigene was excised from the vector and ligated into the eukaryotic expression vectors. SV40-pA consists of the SV40 early promoter linked to exon 1 and part of exon 2 (including the intron) of rabbit β globin to facilitate splicing of any cloned insert. This is of particular importance if the construct is to be used to generate transgenic lines. After a single unique blunt restriction site for cloning inserts into, there is the SV40 small T poly A signal sequence. The SV40 promoter will express the minigene in all tissues. The HSA-pA construct is similar except for the use of the human skeletal α actin promoter and tissue specific regulatory sequences which will direct expression of the minigene product only in skeletal muscle.

Once cloned into the expression vectors the unique HpaI site was used to clone in a PCR generated fragment containing the remainder of the utrophin rod domain. We now have expression vectors containing a truncated and full length utrophin coding sequence.

The unique HpaI restriction site has also been used to clone in a synthetic oligonucleotide coding for the amino acid sequence which is recognised by a specific antibody to the myc protein. This will enable minigene constructs to be localised by virtue of their expression of the myc tag and recognition by the antibody. For utrophin this is a problem as the endogenous gene is expressed in all cell types. The use of the tag will demonstrate the presence of the minigene when delivered in a gene therapy protocol. There are available a number of other tags including the Flag epitope (IBI) and Green Fluorescent Protein (Clontech) which could be used in a similar fashion.

The utrophin minigene generated consists of the same domains and repeats as the dystrophin minigene (FIG. 4). The dystrophin minigene was originally copied in vitro from a naturally occurring dystrophin mutation which gave rise to a mild Becker muscular dystrophy phenotype. It has been used successfully in a number of viral vectors being designed for potential gene therapy routes and in transgenic lines which ameliorate the abnormal muscle phenotype in mdx mice. Thus the utrophin minigene will be suitable for cloning into viral vectors designed for specific tissue expression in potential gene therapy procedures.

Verification of the Integrity of Minigenes

It was important to screen for the maintenance of an open reading frame in the PCR generated clones given the propensity for Taq thermostable polymerase to introduce mutations. The PCR products were cloned into a vector which had RNA polymerase binding sites allowing the cloned insert to be transcribed and translated generating a radiolabelled protein. If expressed proteins were observed of the correct molecular weight it was inferred that the PCR product had no stop mutations. These products were then western blotted to see if they were recognised by utrophin antibodies. A positive result demonstrated that the expressed protein was in the correct frame to generate the epitopes recognised by the antibodies. Ten different clones both for the PCR2.0 and PCR4.0 were screened in this manner. In all cases full length expression was observed. All PCR2.0 and PCR4.0 clones were detected by MANNUT1 [31] (which recognises the actin binding domain) and MANCHO7 [18] (which recognises the latter half of the carboxy-terminal) respectively.

Two minigenes were constructed from two different PCR2.0 and 4.0 clones which met the criteria above and cloned into the expression vectors. To check the integrity of the completed minigene, COS cells were transiently transfected with both SV40-PCR6.0 minigenes (A4 and B1) and harvested after time points. Expression of the PCR6.0 minigene protein was identified by western blotting using MANNUT1 [31] and MANCH07 [18].

Similar transfections were done using these constructs then the cells fixed and immunostained using MANCH07 [18]. Staining of the minigene appeared to be membrane bound suggesting that the actin binding domain or the CRCT or both are functional in order to explain the staining pattern seen.

The myc tag epitope has also been cloned in frame into the unique HpaI site within the minigene. This construct, SV40-PCR6.0-myc, was also transfected into COS cells and immunolocalised using-the myc tag mouse monoclonal antibody, 9 E10. Again membrane localisation was observed showing that introduction of the 10 amino acids which constitutes the myc tag epitope does not appear to effect the properties of the minigene.

EXAMPLE 2 In Vivo Compensation for Dystrophic Deficiency by Utrophin Expression

We have tested expressing a utrophin transgene in the dystrophin deficient mdx mouse. Our results indicate that high expression of the utrophin transgene in skeletal muscle can reverse the dystrophic pathology. These data suggest that systemic up-regulation of utrophin in DMD patients is a very promising avenue for the development of an effective treatment for this devastating disorder.

A truncated utrophin transgene was modeled on the Becker dystrophin transgene which has been shown to correct the dystrophic phenotype of mdx mice ^([5,6]) (FIG. 5A). In order to generate high levels of muscle expression the utrophin transgene was driven by the human skeletal alpha actin (HSA) promoter (FIG. 5B). A number of transgenic lines expressing the utrophin transgene were generated with differing levels of transgenic expression. Immunoblot analysis of muscle samples from transgenic lines demonstrating high level expression are shown in FIG. 5C. The multiple fainter bands are probably due to the proteolytic breakdown of the highly expressed transgene product ^([24]). Line 347 also shows weak expression of the transgene in the heart. Analysis of the F-3 line shows no evidence of transgene expression in heart, brain, kidney, lung, liver, intestine, skin or pancreas was observed. To demonstrate that the utrophin transgene localised to the sarcolemma, immunofluorescence of skeletal muscle sections was performed using utrophin and dystrophin specific antibodies. Examination of the sarcolemmal localisation pattern of dystrophin and the utrophin transgene in consecutive muscle sections demonstrated that they are able to co-localise in vivo. The normal localisation of utrophin in adult skeletal muscle is exclusively at the neuromuscular and myotendenous junctions and in the capillaries and nerves ^([3,31]). Immunostaining of unfixed 8 μm TA muscle cryosections was done with 1/25 dilution of G3 (anti-utrophin) or 1/400 dilution of P6 (anti-dystrophin ^([33])) Initially the sections were blocked in 10% heat inactivated foetal calf serum in 50 mM Tris, 150 mM NaCl pH7.5 (TBS), then the primary antibody diluted in TBS added and incubated for 1 h at room temperature. The slides were washed 4× in TBS for 5 min each then incubated for a further hour at room temperature with 1/1000 dilution FITC conjugated sheep anti-rabbit IgG (Sigma) diluted in TBS. Finally the slides were washed as before, mounted with VectaShield (Vector Labs) and photographed using a Leica DMRBE microscope and photomicrograph system.

Although the dystrophin deficient mdx mouse is only mildly affected, histological and physiological analysis reveals a number of muscle defects in common with DMD patients including muscle fibre degeneration giving rise to a dramatic elevation of serum creatine kinase (CK) and evidence of massive myofibre regeneration with most fibres having centrally located nuclei ^([34]). Thus changes in the levels of serum CK and numbers of centralised nuclei have been used to monitor the pathology of the muscle in a number of transgenic lines expressing dystrophin transgenes in mdx mice ^([5,6,7,24]). Male transgenic F-3 mice carrying the utrophin transgene were crossed with dystrophin deficient female mdx mice and the resultant offspring analysed (FIG. 2A). The CK levels of 5 week old male transgenic mdx mice had fallen to approximately a quarter of the non transgenic mdx male littermates. Females whether transgenic or not have essentially normal levels of serum CK. The reduction in the serum levels of CK in the transgenic male mdx littermates signifies a change in the muscle pathology of these mice and implies that a significant decrease in muscle degeneration has occurred. FIG. 2B shows the contrast in numbers of centralised nuclei in frozen sections from the soleus and tibialis anteria (TA) muscle of transgenic and non-transgenic male mdx mice. The numbers of centrally nucleated myofibres is markedly reduced in the two muscle types examined showing that the amount of fibre regeneration is decreased. The difference in numbers of central nuclei between the transgenic mdx TA (˜10%) and soleus (˜30%) is probably explained by the fact that the HSA promoter is expressed at lower levels in the slow twitch fibres which essentially populate the soleus muscle compared to the fast twitch fibres of the TA. This is an important observation as it implies that the levels of utrophin transgene are important for amelioration of the muscle phenotype.

Dystrophin is normally associated with a large oligomeric protein complex (dystrophin protein complex; DPC) embedded in the sarcolemma ^([3,35]). Loss of dystrophin in DMD patients and mdx mice also results in a dramatic loss of sarcolemmal DPC ^([36]). In transgenic mdx mice expressing the full length and truncated dystrophin transgenes, re-establishment of components of the DPC at the sarcolemma is an important marker for the restoration of muscle strength by dystrophin transgenes ^([5,6,7,24]). We looked at the results of immunostaining for components of the DPC in TA muscle from male mdx or mdx expressing the utrophin transgene. This was as described above. The primary antibodies were goat polyclonal sera to α/β-dystroglycan ^([37]) (FP-B, 1/10), rabbit polyclonal sera to α-sarcoglycan ^([38]) (1/5) and sheep polyclonal sera to γ-sarcoglycan ^([39]) (1/10). FITC conjugated secondary antibodies to goat, rabbit and sheep were diluted 1/50, 1/200 and 1/50 respectively. Sarcolemmal staining of all myofibres by utrophin specific antibody was seen in transgenic muscle. However in the non-transgenic mice there is virtually no sarcolemmal staining apart from neuromuscular junctions and regions likely to contain regenerating fibres. In all cases using polyclonal antibodies specific to α-sarcoglycan, γ-sarcoglycan. and α/β-dystroglycan there was a notable increase in the staining at the sarcolemma of transgenic TA muscle indicating an elevation in correctly localised, sarcolemmal bound DPCs. The increase in sarcolemmal staining of these components in the soleus muscle is greater than the non-transgenic mdx males but not as elevated as in the TA. This result suggests that increased utrophin transgene expression correlates with an increase in sarcolemmal bound DPC.

Analysis of the mdx diaphragm has shown that this muscle exhibits a continued pattern of degeneration, fibrosis and functional deficit throughout the life span of the mdx mouse which is comparable to DMD skeletal muscle ^([40]). Thus for utrophin to be capable of replacing dystrophin, over-expression of utrophin in this muscle has to alter the pathology in a similar way as demonstrated for the dystrophin transgenic mdx mice ^([5,6,7,24]). Immunostaining of diaphragm sections using a utrophin antibody demonstrates the sarcolemmal localisation of the utrophin transgene expressed in the transgenic mdx mouse (utro-tg mdx) compared to the normal and mdx seen is the re-establishment of α-sarcoglycan at the sarcolemma of the transgenic mdx diaphragm at levels similar to the normal diaphragm. Sarcolemmal staining of the transgenic mdx diaphragm similar to normal is also seen using antibodies specific to α/β-dystroglycan and γ-sarcoglycan (data not shown). Thus, as in skeletal muscle, expression of the utrophin transgene in diaphragm relocalises the DPC to the sarcolemma. Histological analysis of haematoxylin and eosin stained sections of mdx diaphragm shows extensive regions of fibrosis, cellular infiltration and variable myofibre size containing centralised nuclei. However the utrophin transgenic diaphragm looks essentially the same as normal, with no necrosis, regular myofibre size and virtually no centralised nuclei. In the mdx diaphragm, even in regions which have no necrosis so appear more histologically normal, on higher magnification the myofibres are still of variable size often containing centralised nuclei which is indicative of continual regeneration. In the utrophin transgenic diaphragm even at higher magnification, the whole muscle appears normal. A return to normal histology and establishment of the DPC are two important observations, as seen with the dystrophin transgenic mice ^([5,6,7,24]), which predicts a major recovery of the utrophin transgenic diaphragm from a dystrophic phenotype.

We have demonstrated a significant decrease in the dystrophic muscle phenotype of mdx mice by expressing a utrophin transgene at high levels in the skeletal muscle and the diaphragm. These results, for the first time, strongly suggest that utrophin can replace dystrophin in vivo This implies that use of small molecules which increase the normal utrophin muscle expression to compensate and therefore alleviate the consequences of a lack of dystrophin is a promising avenue for DMD therapy. This approach would potentially target all muscles and thus prolong life by conserving the respiratory and cardiac muscles. Utrophin is expressed in many tissues so a generalised upregulation may not have detrimental side effects. In our experimental animal model, the normal mice expressing the utrophin transgene at high levels appear to suffer no deleterious side effects in their skeletal and diaphragm muscles. A precedent for such a gene therapy approach using butyrate to upregulate fetal haemoglobin is having success in clinical trials of sickle cell disease ^([41,42]). Only 20-30% of the wild type levels of dystrophin are required to significantly reduce the dystrophic phenotype in mdx mice ^(9,10). It will be interesting to determine whether similar levels of utrophin will be adequate to compensate for dystrophin loss. In addition, since utrophin is normally expressed in all tissues, including muscle, the use of this utrophin transgene rather than a dystrophin transgene in conventional gene therapy approaches e.g. using viruses or liposomes may avert any potential immunological responses against the transgene.

Muscle from transgenic mdx and mdx mice were stressed in vitro. Essentially the test monitors the high mechanical stress produced by force lengthening during active contraction and measures the decrease in force. The method is essentially described in detail by Deconinck et al ^([46]). The measure of deterioration is a decrease in the force a muscle can apply. This force drop is irreversible and correlates with the number of damaged muscle fibres. Mdx muscle is particularly sensitive to this test and deteriorates greatly ^([46]).

Our data demonstrate that the force drop in mdx mice is −55%. However, in the utrophin transgenic littermates the force drop was only −20%. Normal mouse muscle usually has a force drop of −15%. Thus the expression of the utrophin transgene in mdx mice considerably decreases the damage caused by large mechanical stress.

Methods

Transgene Construction and Microinjection

The amino- and carboxy-terminal portions of utrophin were cloned as PCR products using overlapping cDNAs as template then ligated together in-frame to produce the truncated utrophin cDNA. The PCR product was then cloned into a vector containing the 2.2 kb human skeletal α-actin (HSA) promoter and regulatory regions ^([43,44]) and SV40 large T poly A site. The cloning sites were such that the transgene was located near the beginning of the second HSA untranslated exon and the Asp718/NotI sites were used to liberate the complete fragment. Transgenic mice were generated by microinjection of the purified HSA transgene insert into the pronucleus of F₂ hybrid oocytes from C57BL/6xCBA/CA parents ^([45]). Positive transgenic mice were identified by southern blotting using a probe to the central part of the utrophin transgene. A number of founder F₀ males were bred to generate more offspring for analysis and breeding.

Protein Analysis

Total muscle extracts were prepared by homogenisation in 1 ml extraction buffer (75 mM Tris pH6.8, 3.8% SDS, 4M Urea, 20% Glycerol, 5% β-mercaptoethanol) then heated 95° C. for 5 min. Usually 50 μg of total protein (quantitated using Biorad DC protein assay kit) was loaded onto 6. polyacrylamide gels and transferred to nitrocellulose. Utrophin transgene expression was detected using a 1/200 dilution of mouse anti-utrophin monoclonal antibody (MANCHO7 ^([18])) and visualised using anti-mouse IgG-POD and chemiluminescence (Boehringer). For sectioning, skeletal muscle samples were removed and immersed in OCT compound (BDH) and frozen in liquid nitrogen cooled isopentane. Diaphragm was removed, cut in half then rolled longitudinally and sandwiched between Ox liver to facilitate orientation and easier sectioning. The sandwich was then frozen. Immunostaining of unfixed 8 μm cryosections was performed by blocking the sections in 10% heat inactivated foetal calf serum in 50 mM Tris, 150 mM NaCl pH7.5 (TBS), then the primary antibody diluted in TBS added and incubated for 1 h at room temperature. The slides were washed 4× in TBS for 5 min each then incubated for a further hour at room temperature with conjugated second antibody diluted in TBS. Finally the slides were washed as before, mounted with VectaShield (Vector Labs) and photographed using a Leica DMRBE microscope and photomicrograph system.

Antibodies used for Immunofluorescence

Antibodies were used at the following dilutions. Polyclonal rabbit against utrophin (G3, 1/25), dystrophin (P6 ^([33]), 1/400), β1-syntrophin (syn35, 1/50) α-sarcoglycan ^([38]) (1/5). Goat polyclonal against α/β-dystroglycan (FP-B ^([37]), 1/10). FITC conjugated secondary antibody to goat (Sigma) and Cy3 conjugated secondary antibody to rabbit (Jackson Laboratories) were diluted 1/50 and 1/200 respectively.

Creatine Kinase Assay

Serum CK levels from 4-5 week old mice generated from 4 F₃ litters resultant from a male transgenic mouse crossed with female mdx were assayed. The tail tips were cut off and DNA prepared for Southern blotting to establish the transgenic status of each mouse. Blood was collected simultaneously, allowed to clot and serum removed. Serum creatine kinase levels were measured using the Boehringer NAC-CK kit and 5 μl of serum. The rate per minute was averaged over 4 min and calculated as U/l.

EXAMPLE 3

To see if expression of utrophin is beneficial to muscle in the process of regenerating, the myc tagged truncated utrophin minigene under the control of the HSA promoter (HSA-PCR6.0-myc) was directly injected into mdx muscle.

Our data demonstrate the sarcolemmal localisation of the utrophin minigene in a proportion of fibres close to the injection site. The utrophin minigene was detected using the antibody 9E10 which is specific to the myc tag epitope. Importantly where 9E10 was localised there was a significant staining of α- and γ-sarcoglycan. The α-sarcoglycan staining was essentially negative in other fibres.

Re-establishment of the dystrophin protein complex has been shown to be an important marker for muscle recovery ^([5,6,7,24]). This result suggests that even when the disease process has manifested itself, namely the degeneration and regeneration seen in mdx muscle, expression of utrophin is beneficial. This is important when considering that in DMD one third of effected boys are new mutations. Thus only when the first symptoms of DMD manifest themselves after a couple of years after birth can diagnosis be attained.

EXAMPLE 4

Utilising a PCR strategy to generate fragments from human 1 st strand DNA, the remainder of the human utrophin sequences missing from PCR6.0 were cloned. The fragment utilised primers which allowed the rod domain to be cloned into the unique HpaI restriction site (see FIG. 2c) to produce a clone which contained all of the amino acid coding sequence to produce the complete utrophin protein. FIG. 9 shows the DNA sequence of the full length utrophin construct with the amino acid sequence shown above using the standard single letter code.

The utrophin full length construct has been cloned into the human skeletal alpha actin promoter (HSA) expression construct in a similar manner to that shown in FIG. 5b. This full length utrophin expression construct has been used to generate transgenic mice capable of expressing the full length utrophin protein in mouse muscle. Similar experiments may be performed as described in Example 2 to identify any differences in the effectiveness of the full length utrophin protein compared with the truncated utrophin protein in alleviating the muscle pathology in mdx mice.

In order to assess whether high levels of utrophin expression in all tissues is detrimental, to assist in planning therapeutic protocols and in particular choosing between tissue-specific expression or non-specific expression, a mouse model is being developed using the full length utrophin construct. Transgenic mice will be created expressing the full length utrophin protein under the regulation of a promoter which is expressed in all tissues. The promoter chosen for the first experiments is the human Ubiquitin-C promoter which has been shown to express in all tissues. Once these mice are shown to be expressing the full length utrophin transgene they will be monitored to identify any potential side effects caused by abnormally high levels of utrophin.

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15 1 710 DNA Mus sp. 1 atggccaagt atggggacct tgaagcctgg cctgatgggc agatcgaatt cagtgacatc 60 attgagtcca gatctgatga acacaatgat gtacagaaga aaacctttac caaatggata 120 aacgctcgat tttccaagag tgggaaacca cccatcagtg atatgttctc agacctcaaa 180 gatgggagaa agctcttgga tcttctcgaa ggcctcacag gaacatcatt gccaaaggaa 240 cgtggttcca caagggtgca tgccttaaac aatgtcaacc gagtgctaca ggttttacat 300 cagaacaatg tggacttggt gaatattgga ggcacggaca ttgtggatgg aaatcccaag 360 ctgactttag ggttactctg gagcatcatt ctgcactggc aggtgaagga tgtcatgaaa 420 gatatcatgt cagacctgca gcagacaaac agcgagaaga tcctgctgag ctgggtgcgg 480 cagaccacca ggccctacag tcaagtcaac gtcctcaact tcaccaccag ctggaccgat 540 ggactcgcgt tcaacgccgt gctccaccgg cacaaaccag atctcttcag ctgggacaga 600 gtggtcaaaa tgtccccaat tgagagactt gaacatgctt ttagcaaggc ccacacttat 660 ttgggaattg aaaagcttct agatcctgaa gatgttgctg tgcatctccc 710 2 713 DNA Rattus sp. 2 atggccaagt acgggcacct tgaagccagc cctgatgatg ggcagaatca gttcagtgat 60 atcattaagt ccagatctga tgaacacaac gatgtacaga agaaaacctt taccaaatgg 120 ataaacgctc gattttcaaa gagtgggaaa ccacccatca atgatatgtt ctcagacctc 180 aaagatggga gaaagctctt ggatcttcta gaaggcctca caggaacatc attgccaaag 240 gaacgtggtt ccacaagggt gcatgcctta aataatgtca accgggtgct gcaggtttta 300 catcagaaca atgtggagct ggtgaatatc ggaggcactg acattgtgga tggaaatccc 360 aagctgacgc tgggtttgct gtggagtatt attctgcact ggcaggtgaa ggatgtcatg 420 aaagatatca tgtcagacct gcagcagacg aacagcgaga agatcctgct gagctgggta 480 cggcagacca ccaggcccta cagccaagtc aacgtcctca acttcaccac cagctggaca 540 gatggactcg cattcaacgc cgtgctccac cggcacaaac cagatctctt cagctgggac 600 agagtggtca aaatgtcccc aactgagaga cttgaacatg cttttagcaa ggcccatact 660 tatttgggga ttgaaaaact tctggatcct gaagatgttg ccgtgcagct ccc 713 3 713 DNA Homo sapiens 3 atggccaagt atggagaaca tgaagccagt cctgacaatg ggcagaacga attcagtgat 60 atcattaagt ccagatctga tgaacacaat gacgtacaga agaaaacctt taccaaatgg 120 ataaatgctc gattttcaaa gagtgggaaa ccacccatca atgatatgtt cacagacctc 180 aaagatggaa ggaagctatt ggatcttcta gaaggcctca caggaacatc actgccaaag 240 gaacgtggtt ccacaagggt acatgcctta aataacgtca acagagtgct gcaggtttta 300 catcagaaca atgtggaatt agtgaatata gggggaactg acattgtgga tggaaatcac 360 aaactgactt tggggttact ttggagcatc attttgcact ggcaggtgaa agatgtcatg 420 aaggatgtca tgtcggacct gcagcagacg aacagtgaga agatcctgct cagctgggtg 480 cgtcagacca ccaggcccta cagccaagtc aacgtcctca acttcaccac cagctggaca 540 gatggactcg cctttaatgc tgtcctccac cgacataaac ctgatctctt cagctgggat 600 aaagttgtca aaatgtcacc aattgagaga cttgaacatg ccttcagcaa ggctcaaact 660 tatttgggaa ttgaaaagct gttagatcct gaagatgttg ccgttcggct tcc 713 4 200 DNA Mus sp. 4 tgatgacctg ccctccctgc agaagctgct tcaagaacat aaaagtttgc aaaatgacct 60 tgaagctgaa caggtgaagg taaattcctt aactcacatg gtggtgattg tggatgaaaa 120 cagtggggag agtgccacag ctcttctgga agatcagtta cagaaactgg gtgagcgctg 180 gacagctgta tgccgctgga 200 5 200 DNA Rattus sp. 5 tgacgaccta ccctccctgc aaaacctgct tgaagaacat aaaagtttgc aaagtgacct 60 cgaagctgag caggtgaagg tgaattcctt aactcatatg gtggtgattg tggatgaaaa 120 cagtggggag agcgccacag ctgttttgga agatcagtta cagaaactgg gtgagcgctg 180 gacagctgta tgccgctgga 200 6 200 DNA Homo sapiens 6 tgatgatgtg aaatctctac aaaagctgct agaagaacat aaaagtttgc aaagtgatct 60 tgaggctgaa caggtgaaag taaattcact aactcacatg gtggtcattg ttgatgaaaa 120 cagtggtgag agcgctacag ctatcctaga agaccagtta cagaaacttg gtgagcgctg 180 gacagcagta tgccgttgga 200 7 6045 DNA Artificial Sequence CDS (11)..(6037) Description of Artificial Sequence Chimeric 7 actagtcaag atg gcc aag tat gga gaa cat gaa gcc agt cct gac aat 49 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn 1 5 10 ggg cag aac gaa ttc agt gac atc att gag tcc aga tct gat gaa cac 97 Gly Gln Asn Glu Phe Ser Asp Ile Ile Glu Ser Arg Ser Asp Glu His 15 20 25 aat gat gta cag aag aaa acc ttt acc aaa tgg ata aac gct cga ttt 145 Asn Asp Val Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe 30 35 40 45 tcc aag agt ggg aaa cca ccc atc agt gat atg ttc tca gac ctc aaa 193 Ser Lys Ser Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys 50 55 60 gat ggg aga aag ctc ttg gat ctt ctc gaa ggc ctc aca gga aca tca 241 Asp Gly Arg Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser 65 70 75 ttg cca aag gaa cgt ggt tcc aca agg gtg cat gcc tta aac aat gtc 289 Leu Pro Lys Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val 80 85 90 aac cga gtg cta cag gtt tta cat cag aac aat gtg gac ttg gtg aat 337 Asn Arg Val Leu Gln Val Leu His Gln Asn Asn Val Asp Leu Val Asn 95 100 105 att gga ggc acg gac att gtg gat gga aat ccc aag ctg act tta ggg 385 Ile Gly Gly Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly 110 115 120 125 tta ctc tgg agc atc att ctg cac tgg cag gtg aag gat gtc atg aaa 433 Leu Leu Trp Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys 130 135 140 gat atc atg tca gac ctg cag cag aca aac agc gag aag atc ctg ctg 481 Asp Ile Met Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu 145 150 155 agc tgg gtg cgg cag acc acc agg ccc tac agt caa gtc aac gtc ctc 529 Ser Trp Val Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu 160 165 170 aac ttc acc acc agc tgg acc gat gga ctc gcg ttc aac gcc gtg ctc 577 Asn Phe Thr Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu 175 180 185 cac cgg cac aaa cca gat ctc ttc agc tgg gac aga gtg gtc aaa atg 625 His Arg His Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met 190 195 200 205 tcc cca att gag aga ctt gaa cat gct ttt agc aag gcc cac act tat 673 Ser Pro Ile Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr 210 215 220 ttg gga att gaa aag ctt cta gat cct gaa gat gtt gct gtg cat ctc 721 Leu Gly Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val His Leu 225 230 235 ccn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn ncc gtt gag gtg 769 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Glu Val 240 245 250 ctt cct cag caa gtc acg ata gat gcc atc cga gag gtg gag act ctc 817 Leu Pro Gln Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu 255 260 265 cca agg aag tat aag aaa gaa tgt gaa gag gaa gaa att cat atc cag 865 Pro Arg Lys Tyr Lys Lys Glu Cys Glu Glu Glu Glu Ile His Ile Gln 270 275 280 285 agt gca gtg ctg gca gag gaa ggc cag agt ccc cga gct gag acc cct 913 Ser Ala Val Leu Ala Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr Pro 290 295 300 agc acc gtc act gaa gtg gac atg gat ttg gac agc tac cag ata gcg 961 Ser Thr Val Thr Glu Val Asp Met Asp Leu Asp Ser Tyr Gln Ile Ala 305 310 315 cta gag gaa gtg ctg acg tgg ctg ctg tcc gcg gag gac acg ttc cag 1009 Leu Glu Glu Val Leu Thr Trp Leu Leu Ser Ala Glu Asp Thr Phe Gln 320 325 330 gag caa gat gac att tct gat gat gtc gaa gaa gtc aaa gag cag ttt 1057 Glu Gln Asp Asp Ile Ser Asp Asp Val Glu Glu Val Lys Glu Gln Phe 335 340 345 gct acc cat gaa act ttt atg atg gag ctg aca gca cac cag agc agc 1105 Ala Thr His Glu Thr Phe Met Met Glu Leu Thr Ala His Gln Ser Ser 350 355 360 365 gtg ggg agc gtc ctg cag gct ggc aac cag ctg atg aca caa ggg act 1153 Val Gly Ser Val Leu Gln Ala Gly Asn Gln Leu Met Thr Gln Gly Thr 370 375 380 ctg tca gag gag gag gag ttt gag atc cag gaa cag atg acc ttg ctg 1201 Leu Ser Glu Glu Glu Glu Phe Glu Ile Gln Glu Gln Met Thr Leu Leu 385 390 395 aat gca agg tgg gag gcg ctc cgg gtg gag agc atg gag agg cag tcc 1249 Asn Ala Arg Trp Glu Ala Leu Arg Val Glu Ser Met Glu Arg Gln Ser 400 405 410 cgg ctg cac gac gct ctg atg gag ctg cag aag aaa cag ctg cag cag 1297 Arg Leu His Asp Ala Leu Met Glu Leu Gln Lys Lys Gln Leu Gln Gln 415 420 425 ctc tca agc tgg ctg gcc ctc aca gaa gag cgc att cag aag atg gag 1345 Leu Ser Ser Trp Leu Ala Leu Thr Glu Glu Arg Ile Gln Lys Met Glu 430 435 440 445 agc cct ccg ctg ggt gat gac ctg ccc tcc ctg cag aag ctg ctt caa 1393 Ser Pro Pro Leu Gly Asp Asp Leu Pro Ser Leu Gln Lys Leu Leu Gln 450 455 460 gaa cat aaa agt ttg caa aat gac ctt gaa gct gaa cag gtg aag gta 1441 Glu His Lys Ser Leu Gln Asn Asp Leu Glu Ala Glu Gln Val Lys Val 465 470 475 aat tcc tta act cac atg gtg gtg att gtg gat gaa aac agt ggg gag 1489 Asn Ser Leu Thr His Met Val Val Ile Val Asp Glu Asn Ser Gly Glu 480 485 490 agt gcc aca gct ctt ctg gaa gat cag tta cag aaa ctg ggt gag cgc 1537 Ser Ala Thr Ala Leu Leu Glu Asp Gln Leu Gln Lys Leu Gly Glu Arg 495 500 505 tgg aca gct gta tgc cgc tgg act gaa gaa cgt tgg aac agg ttg caa 1585 Trp Thr Ala Val Cys Arg Trp Thr Glu Glu Arg Trp Asn Arg Leu Gln 510 515 520 525 gaa atc agt att ctg tgg cag gaa tta ttg gaa gag cag tgt ctg ttg 1633 Glu Ile Ser Ile Leu Trp Gln Glu Leu Leu Glu Glu Gln Cys Leu Leu 530 535 540 gag gct tgg ctc acc gaa aag gaa gag gct ttg aat aaa gtt caa acc 1681 Glu Ala Trp Leu Thr Glu Lys Glu Glu Ala Leu Asn Lys Val Gln Thr 545 550 555 agc aac ttt aaa gac cag aag gaa cta agt gtc agt gtc cgg cgt ctg 1729 Ser Asn Phe Lys Asp Gln Lys Glu Leu Ser Val Ser Val Arg Arg Leu 560 565 570 gct ata ttg aag gaa gac atg gaa atg aag agg cag act ctg gat caa 1777 Ala Ile Leu Lys Glu Asp Met Glu Met Lys Arg Gln Thr Leu Asp Gln 575 580 585 ctg agt gag att ggc cag gat gtg ggc caa tta ctc agt aat ccc aag 1825 Leu Ser Glu Ile Gly Gln Asp Val Gly Gln Leu Leu Ser Asn Pro Lys 590 595 600 605 gca tct aag aag atg aac agt gac tct gag gag cta aca cag aga tgg 1873 Ala Ser Lys Lys Met Asn Ser Asp Ser Glu Glu Leu Thr Gln Arg Trp 610 615 620 gat tct ctg gtt cag aga ctc gaa gac tct tct aac cag gtg act cag 1921 Asp Ser Leu Val Gln Arg Leu Glu Asp Ser Ser Asn Gln Val Thr Gln 625 630 635 gcg gta gcg aag ctc ggc atg tcc cag att cca cag aag gac cta ttg 1969 Ala Val Ala Lys Leu Gly Met Ser Gln Ile Pro Gln Lys Asp Leu Leu 640 645 650 gag acc gtt cat gtg aga gaa aaa ggg atg gtg aag aag ccc aag cag 2017 Glu Thr Val His Val Arg Glu Lys Gly Met Val Lys Lys Pro Lys Gln 655 660 665 gaa ctg cct cct ccg tta aca aag gct gag cat gct atg caa aag aga 2065 Glu Leu Pro Pro Pro Leu Thr Lys Ala Glu His Ala Met Gln Lys Arg 670 675 680 685 tca acc acc gaa ttg gga gaa aac ctg caa gaa tta aga gac tta act 2113 Ser Thr Thr Glu Leu Gly Glu Asn Leu Gln Glu Leu Arg Asp Leu Thr 690 695 700 caa gaa atg gaa gta cat gct gaa aaa ctc aaa tgg ctg aat aga act 2161 Gln Glu Met Glu Val His Ala Glu Lys Leu Lys Trp Leu Asn Arg Thr 705 710 715 gaa ttg gag atg ctt tca gat aaa agt ctg agt tta cct gaa agg gat 2209 Glu Leu Glu Met Leu Ser Asp Lys Ser Leu Ser Leu Pro Glu Arg Asp 720 725 730 aaa att tca gaa agc tta agg act gta aat atg aca tgg aat aag att 2257 Lys Ile Ser Glu Ser Leu Arg Thr Val Asn Met Thr Trp Asn Lys Ile 735 740 745 tgc aga gag gtg cct acc acc ctg aag gaa tgc atc cag gag ccc agt 2305 Cys Arg Glu Val Pro Thr Thr Leu Lys Glu Cys Ile Gln Glu Pro Ser 750 755 760 765 tct gtt tca cag aca agg att gct gct cat cct aat gtc caa aag gtg 2353 Ser Val Ser Gln Thr Arg Ile Ala Ala His Pro Asn Val Gln Lys Val 770 775 780 gtg cta gta tca tct gcg tca gat att cct gtt cag tct cat cgt act 2401 Val Leu Val Ser Ser Ala Ser Asp Ile Pro Val Gln Ser His Arg Thr 785 790 795 tcg gaa att tca att cct gct gat ctt gat aaa act ata aca gaa cta 2449 Ser Glu Ile Ser Ile Pro Ala Asp Leu Asp Lys Thr Ile Thr Glu Leu 800 805 810 gcc gac tgg ctg gta tta atc gac cag atg ctg aag tcc aac att gtc 2497 Ala Asp Trp Leu Val Leu Ile Asp Gln Met Leu Lys Ser Asn Ile Val 815 820 825 act gtt ggg gat gta gaa gag atc aat aag acc gtt tcc cga atg aaa 2545 Thr Val Gly Asp Val Glu Glu Ile Asn Lys Thr Val Ser Arg Met Lys 830 835 840 845 att aca aag gct gac tta gaa cag cgc cat cct cag ctg gat tat gtt 2593 Ile Thr Lys Ala Asp Leu Glu Gln Arg His Pro Gln Leu Asp Tyr Val 850 855 860 ttt aca ttg gca cag aat ttg aaa aat aaa gct tcc agt tca gat atg 2641 Phe Thr Leu Ala Gln Asn Leu Lys Asn Lys Ala Ser Ser Ser Asp Met 865 870 875 aga aca gca att aca gaa aaa ttg gaa agg gtc aag aac cag tgg gat 2689 Arg Thr Ala Ile Thr Glu Lys Leu Glu Arg Val Lys Asn Gln Trp Asp 880 885 890 ggc acc cag cat ggc gtt gag cta aga cag cag cag ctt gag gac atg 2737 Gly Thr Gln His Gly Val Glu Leu Arg Gln Gln Gln Leu Glu Asp Met 895 900 905 att att gac agt ctt cag tgg gat gac cat agg gag gag act gaa gaa 2785 Ile Ile Asp Ser Leu Gln Trp Asp Asp His Arg Glu Glu Thr Glu Glu 910 915 920 925 ctg atg aga aaa tat gag gct cga ctc tat att ctt cag caa gcc cga 2833 Leu Met Arg Lys Tyr Glu Ala Arg Leu Tyr Ile Leu Gln Gln Ala Arg 930 935 940 cgg gat cca ctc acc aaa caa att tct gat aac caa ata ctg ctt caa 2881 Arg Asp Pro Leu Thr Lys Gln Ile Ser Asp Asn Gln Ile Leu Leu Gln 945 950 955 gaa ctg ggt cct gga gat ggt atc gtc atg gcg ttc gat aac gtc ctg 2929 Glu Leu Gly Pro Gly Asp Gly Ile Val Met Ala Phe Asp Asn Val Leu 960 965 970 cag aaa ctc ctg gag gaa tat ggg agt gat gac aca agg aat gtg aaa 2977 Gln Lys Leu Leu Glu Glu Tyr Gly Ser Asp Asp Thr Arg Asn Val Lys 975 980 985 gaa acc aca gag tac tta aaa aca tca tgg atc aat ctc aaa caa agt 3025 Glu Thr Thr Glu Tyr Leu Lys Thr Ser Trp Ile Asn Leu Lys Gln Ser 990 995 1000 1005 att gct gac aga cag aac gcc ttg gag gct gag tgg agg acg gtg cag 3073 Ile Ala Asp Arg Gln Asn Ala Leu Glu Ala Glu Trp Arg Thr Val Gln 1010 1015 1020 gcc tct cgc aga gat ctg gaa aac ttc ctg aag tgg atc caa gaa gca 3121 Ala Ser Arg Arg Asp Leu Glu Asn Phe Leu Lys Trp Ile Gln Glu Ala 1025 1030 1035 gag acc aca gtg aat gtg ctt gtg gat gcc tct cat cgg gag aat gct 3169 Glu Thr Thr Val Asn Val Leu Val Asp Ala Ser His Arg Glu Asn Ala 1040 1045 1050 ctt cag gat agt atc ttg gcc agg gaa ctc aaa cag cag atg cag gac 3217 Leu Gln Asp Ser Ile Leu Ala Arg Glu Leu Lys Gln Gln Met Gln Asp 1055 1060 1065 atc cag gca gaa att gat gcc cac aat gac ata ttt aaa agc att gac 3265 Ile Gln Ala Glu Ile Asp Ala His Asn Asp Ile Phe Lys Ser Ile Asp 1070 1075 1080 1085 gga aac agg cag aag atg gta aaa gct ttg gga aat tct gaa gag gct 3313 Gly Asn Arg Gln Lys Met Val Lys Ala Leu Gly Asn Ser Glu Glu Ala 1090 1095 1100 act atg ctt caa cat cga ctg gat gat atg aac caa aga tgg aat gac 3361 Thr Met Leu Gln His Arg Leu Asp Asp Met Asn Gln Arg Trp Asn Asp 1105 1110 1115 tta aaa gca aaa tct gct agc atc agg gcc cat ttg gag gcc agc gct 3409 Leu Lys Ala Lys Ser Ala Ser Ile Arg Ala His Leu Glu Ala Ser Ala 1120 1125 1130 gag aag tgg aac agg ttg ctg atg tcc tta gaa gaa ctg atc aaa tgg 3457 Glu Lys Trp Asn Arg Leu Leu Met Ser Leu Glu Glu Leu Ile Lys Trp 1135 1140 1145 ctg aat atg aaa gat gaa gag ctt aag aaa caa atg cct att gga gga 3505 Leu Asn Met Lys Asp Glu Glu Leu Lys Lys Gln Met Pro Ile Gly Gly 1150 1155 1160 1165 gat gtt cca gcc tta cag ctc cag tat gac cat tgt aag gcc ctg aga 3553 Asp Val Pro Ala Leu Gln Leu Gln Tyr Asp His Cys Lys Ala Leu Arg 1170 1175 1180 cgg gag tta aag gag aaa gaa tat tct gtc ctg aat gct gtc gac cag 3601 Arg Glu Leu Lys Glu Lys Glu Tyr Ser Val Leu Asn Ala Val Asp Gln 1185 1190 1195 gcc cga gtt ttc ttg gct gat cag cca att gag gcc cct gaa gag cca 3649 Ala Arg Val Phe Leu Ala Asp Gln Pro Ile Glu Ala Pro Glu Glu Pro 1200 1205 1210 aga aga aac cta caa tca aaa aca gaa tta act cct gag gag aga gcc 3697 Arg Arg Asn Leu Gln Ser Lys Thr Glu Leu Thr Pro Glu Glu Arg Ala 1215 1220 1225 caa aag att gcc aaa gcc atg cgc aaa cag tct tct gaa gtc aaa gaa 3745 Gln Lys Ile Ala Lys Ala Met Arg Lys Gln Ser Ser Glu Val Lys Glu 1230 1235 1240 1245 aaa tgg gaa agt cta aat gct gta act agc aat tgg caa aag caa gtg 3793 Lys Trp Glu Ser Leu Asn Ala Val Thr Ser Asn Trp Gln Lys Gln Val 1250 1255 1260 gac aag gca ttg gag aaa ctc aga gac ctg cag gga gct atg gat gac 3841 Asp Lys Ala Leu Glu Lys Leu Arg Asp Leu Gln Gly Ala Met Asp Asp 1265 1270 1275 ctg gac gct gac atg aag gag gca gag tcc gtg cgg aat ggc tgg aag 3889 Leu Asp Ala Asp Met Lys Glu Ala Glu Ser Val Arg Asn Gly Trp Lys 1280 1285 1290 ccc gtg gga gac tta ctc att gac tcg ctg cag gat cac att gaa aaa 3937 Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His Ile Glu Lys 1295 1300 1305 atc atg gca ttt aga gaa gaa att gca cca atc aac ttt aaa gtt aaa 3985 Ile Met Ala Phe Arg Glu Glu Ile Ala Pro Ile Asn Phe Lys Val Lys 1310 1315 1320 1325 acg gtg aat gat tta tcc agt cag ctg tct cca ctt gac ctg cat ccc 4033 Thr Val Asn Asp Leu Ser Ser Gln Leu Ser Pro Leu Asp Leu His Pro 1330 1335 1340 tct cta aag atg tct cgc cag cta gat gac ctt aat atg cga tgg aaa 4081 Ser Leu Lys Met Ser Arg Gln Leu Asp Asp Leu Asn Met Arg Trp Lys 1345 1350 1355 ctt tta cag gtt tct gtg gat gat cgc ctt aaa cag ctt cag gaa gcc 4129 Leu Leu Gln Val Ser Val Asp Asp Arg Leu Lys Gln Leu Gln Glu Ala 1360 1365 1370 cac aga gat ttt gga cca tcc tct cag cat ttt ctc tct acg tca gtc 4177 His Arg Asp Phe Gly Pro Ser Ser Gln His Phe Leu Ser Thr Ser Val 1375 1380 1385 cag ctg ccg tgg caa aga tcc att tca cat aat aaa gtg ccc tat tac 4225 Gln Leu Pro Trp Gln Arg Ser Ile Ser His Asn Lys Val Pro Tyr Tyr 1390 1395 1400 1405 atc aac cat caa aca cag acc acc tgt tgg gac cat cct aaa atg acc 4273 Ile Asn His Gln Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met Thr 1410 1415 1420 gaa ctc ttt caa tcc ctt gct gac ctg aat aat gta cgt ttt tct gcc 4321 Glu Leu Phe Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser Ala 1425 1430 1435 tac cgt aca gca atc aaa atc cga aga cta caa aaa gca cta tgt ttg 4369 Tyr Arg Thr Ala Ile Lys Ile Arg Arg Leu Gln Lys Ala Leu Cys Leu 1440 1445 1450 gat ctc tta gag ttg agt aca aca aat gaa att ttc aaa cag cac aag 4417 Asp Leu Leu Glu Leu Ser Thr Thr Asn Glu Ile Phe Lys Gln His Lys 1455 1460 1465 ttg aac caa aat gac cag ctc ctc agt gtt cca gat gtc atc aac tgt 4465 Leu Asn Gln Asn Asp Gln Leu Leu Ser Val Pro Asp Val Ile Asn Cys 1470 1475 1480 1485 ctg aca aca act tat gat gga ctt gag caa atg cat aag gac ctg gtc 4513 Leu Thr Thr Thr Tyr Asp Gly Leu Glu Gln Met His Lys Asp Leu Val 1490 1495 1500 aac gtt cca ctc tgt gtt gat atg tgt ctc aat tgg ttg ctc aat gtc 4561 Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val 1505 1510 1515 tat gac acg ggt cga act gga aaa att aga gtg cag agt ctg aag att 4609 Tyr Asp Thr Gly Arg Thr Gly Lys Ile Arg Val Gln Ser Leu Lys Ile 1520 1525 1530 gga tta atg tct ctc tcc aaa ggt ctc ttg gaa gaa aaa tac aga tat 4657 Gly Leu Met Ser Leu Ser Lys Gly Leu Leu Glu Glu Lys Tyr Arg Tyr 1535 1540 1545 ctc ttt aag gaa gtt gcg ggg ccg aca gaa atg tgt gac cag agg cag 4705 Leu Phe Lys Glu Val Ala Gly Pro Thr Glu Met Cys Asp Gln Arg Gln 1550 1555 1560 1565 ctg ggc ctg tta ctt cat gat gcc atc cag atc ccc cgg cag cta ggt 4753 Leu Gly Leu Leu Leu His Asp Ala Ile Gln Ile Pro Arg Gln Leu Gly 1570 1575 1580 gaa gta gca gct ttt gga ggc agt aat att gag cct agt gtt cgc agc 4801 Glu Val Ala Ala Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser 1585 1590 1595 tgc ttc caa cag aat aac aat aaa cca gaa ata agt gtg aaa gag ttt 4849 Cys Phe Gln Gln Asn Asn Asn Lys Pro Glu Ile Ser Val Lys Glu Phe 1600 1605 1610 ata gat tgg atg cat ttg gaa cca cag tcc atg gtt tgg ctc cca gtt 4897 Ile Asp Trp Met His Leu Glu Pro Gln Ser Met Val Trp Leu Pro Val 1615 1620 1625 tta cat cga gtg gca gca gcg gag act gca aaa cat cag gcc aaa tgc 4945 Leu His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys 1630 1635 1640 1645 aac atc tgt aaa gaa tgt cca att gtc ggg ttc agg tat aga agc ctt 4993 Asn Ile Cys Lys Glu Cys Pro Ile Val Gly Phe Arg Tyr Arg Ser Leu 1650 1655 1660 aag cat ttt aac tat gat gtc tgc cag agt tgt ttc ttt tcg ggt cga 5041 Lys His Phe Asn Tyr Asp Val Cys Gln Ser Cys Phe Phe Ser Gly Arg 1665 1670 1675 aca gca aaa ggt cac aaa tta cat tac cca atg gtg gaa tat tgt ata 5089 Thr Ala Lys Gly His Lys Leu His Tyr Pro Met Val Glu Tyr Cys Ile 1680 1685 1690 cct aca aca tct ggg gaa gat gta cga gac ttc aca aag gta ctt aag 5137 Pro Thr Thr Ser Gly Glu Asp Val Arg Asp Phe Thr Lys Val Leu Lys 1695 1700 1705 aac aag ttc agg tcg aag aag tac ttt gcc aaa cac cct cga ctt ggt 5185 Asn Lys Phe Arg Ser Lys Lys Tyr Phe Ala Lys His Pro Arg Leu Gly 1710 1715 1720 1725 tac ctg cct gtc cag aca gtt ctt gaa ggt gac aac tta gag act cct 5233 Tyr Leu Pro Val Gln Thr Val Leu Glu Gly Asp Asn Leu Glu Thr Pro 1730 1735 1740 atc aca ctc atc agt atg tgg cca gag cac tat gac ccc tca caa tct 5281 Ile Thr Leu Ile Ser Met Trp Pro Glu His Tyr Asp Pro Ser Gln Ser 1745 1750 1755 cct caa ctg ttt cat gat gac acc cat tca aga ata gaa caa tat gcc 5329 Pro Gln Leu Phe His Asp Asp Thr His Ser Arg Ile Glu Gln Tyr Ala 1760 1765 1770 aca cga ctg gcc cag atg gaa agg act aat ggg tct ttt ctc act gat 5377 Thr Arg Leu Ala Gln Met Glu Arg Thr Asn Gly Ser Phe Leu Thr Asp 1775 1780 1785 agc agc tcc acc aca gga agt gtg gaa gac gag cac gcc ctc atc cag 5425 Ser Ser Ser Thr Thr Gly Ser Val Glu Asp Glu His Ala Leu Ile Gln 1790 1795 1800 1805 cag tat tgc caa aca ctc gga gga gag tcc cca gtg agc cag ccg cag 5473 Gln Tyr Cys Gln Thr Leu Gly Gly Glu Ser Pro Val Ser Gln Pro Gln 1810 1815 1820 agc cca gct cag atc ctg aag tca gta gag agg gaa gaa cgt gga gaa 5521 Ser Pro Ala Gln Ile Leu Lys Ser Val Glu Arg Glu Glu Arg Gly Glu 1825 1830 1835 ctg gag agg atc att gct gac ctg gag gaa gaa caa aga aat cta cag 5569 Leu Glu Arg Ile Ile Ala Asp Leu Glu Glu Glu Gln Arg Asn Leu Gln 1840 1845 1850 gtg gag tat gag cag ctg aag gac cag cac ctc cga agg ggg ctc cct 5617 Val Glu Tyr Glu Gln Leu Lys Asp Gln His Leu Arg Arg Gly Leu Pro 1855 1860 1865 gtc ggt tca ccg cca gag tcg att ata tct ccc cat cac acg tct gag 5665 Val Gly Ser Pro Pro Glu Ser Ile Ile Ser Pro His His Thr Ser Glu 1870 1875 1880 1885 gat tca gaa ctt ata gca gaa gca aaa ctc ctc agg cag cac aaa ggt 5713 Asp Ser Glu Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly 1890 1895 1900 cgg ctg gag gct agg atg cag att tta gaa gat cac aat aaa cag ctg 5761 Arg Leu Glu Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu 1905 1910 1915 gag tct cag ctc cac cgc ctc cga cag ctg ctg gag cag cct gaa tct 5809 Glu Ser Gln Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Glu Ser 1920 1925 1930 gat tcc cga atc aat ggt gtt tcc cca tgg gct tct cct cag cat tct 5857 Asp Ser Arg Ile Asn Gly Val Ser Pro Trp Ala Ser Pro Gln His Ser 1935 1940 1945 gca ctg agc tac tcg ctt gat cca gat gcc tcc ggc cca cag ttc cac 5905 Ala Leu Ser Tyr Ser Leu Asp Pro Asp Ala Ser Gly Pro Gln Phe His 1950 1955 1960 1965 cag gca gcg gga gag gac ctg ctg gcc cca ccg cac gac acc agc acg 5953 Gln Ala Ala Gly Glu Asp Leu Leu Ala Pro Pro His Asp Thr Ser Thr 1970 1975 1980 gat ctc acg gag gtc atg gag cag att cac agc acg ttt cca tct tgc 6001 Asp Leu Thr Glu Val Met Glu Gln Ile His Ser Thr Phe Pro Ser Cys 1985 1990 1995 tgc cca aat gtt ccc agc agg cca cag gca atg taa tcactagt 6045 Cys Pro Asn Val Pro Ser Arg Pro Gln Ala Met 2000 2005 8 2008 PRT Artificial Sequence misc_feature (239) ... (250) Description of Artificial Sequence Chimeric; Xaa = Unknown 8 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn Gly Gln Asn 1 5 10 15 Glu Phe Ser Asp Ile Ile Glu Ser Arg Ser Asp Glu His Asn Asp Val 20 25 30 Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe Ser Lys Ser 35 40 45 Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys Asp Gly Arg 50 55 60 Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser Leu Pro Lys 65 70 75 80 Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Arg Val 85 90 95 Leu Gln Val Leu His Gln Asn Asn Val Asp Leu Val Asn Ile Gly Gly 100 105 110 Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly Leu Leu Trp 115 120 125 Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys Asp Ile Met 130 135 140 Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 145 150 155 160 Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu Asn Phe Thr 165 170 175 Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu His Arg His 180 185 190 Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met Ser Pro Ile 195 200 205 Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr Leu Gly Ile 210 215 220 Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val His Leu Pro Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Glu Val Leu Pro Gln 245 250 255 Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu Pro Arg Lys 260 265 270 Tyr Lys Lys Glu Cys Glu Glu Glu Glu Ile His Ile Gln Ser Ala Val 275 280 285 Leu Ala Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr Pro Ser Thr Val 290 295 300 Thr Glu Val Asp Met Asp Leu Asp Ser Tyr Gln Ile Ala Leu Glu Glu 305 310 315 320 Val Leu Thr Trp Leu Leu Ser Ala Glu Asp Thr Phe Gln Glu Gln Asp 325 330 335 Asp Ile Ser Asp Asp Val Glu Glu Val Lys Glu Gln Phe Ala Thr His 340 345 350 Glu Thr Phe Met Met Glu Leu Thr Ala His Gln Ser Ser Val Gly Ser 355 360 365 Val Leu Gln Ala Gly Asn Gln Leu Met Thr Gln Gly Thr Leu Ser Glu 370 375 380 Glu Glu Glu Phe Glu Ile Gln Glu Gln Met Thr Leu Leu Asn Ala Arg 385 390 395 400 Trp Glu Ala Leu Arg Val Glu Ser Met Glu Arg Gln Ser Arg Leu His 405 410 415 Asp Ala Leu Met Glu Leu Gln Lys Lys Gln Leu Gln Gln Leu Ser Ser 420 425 430 Trp Leu Ala Leu Thr Glu Glu Arg Ile Gln Lys Met Glu Ser Pro Pro 435 440 445 Leu Gly Asp Asp Leu Pro Ser Leu Gln Lys Leu Leu Gln Glu His Lys 450 455 460 Ser Leu Gln Asn Asp Leu Glu Ala Glu Gln Val Lys Val Asn Ser Leu 465 470 475 480 Thr His Met Val Val Ile Val Asp Glu Asn Ser Gly Glu Ser Ala Thr 485 490 495 Ala Leu Leu Glu Asp Gln Leu Gln Lys Leu Gly Glu Arg Trp Thr Ala 500 505 510 Val Cys Arg Trp Thr Glu Glu Arg Trp Asn Arg Leu Gln Glu Ile Ser 515 520 525 Ile Leu Trp Gln Glu Leu Leu Glu Glu Gln Cys Leu Leu Glu Ala Trp 530 535 540 Leu Thr Glu Lys Glu Glu Ala Leu Asn Lys Val Gln Thr Ser Asn Phe 545 550 555 560 Lys Asp Gln Lys Glu Leu Ser Val Ser Val Arg Arg Leu Ala Ile Leu 565 570 575 Lys Glu Asp Met Glu Met Lys Arg Gln Thr Leu Asp Gln Leu Ser Glu 580 585 590 Ile Gly Gln Asp Val Gly Gln Leu Leu Ser Asn Pro Lys Ala Ser Lys 595 600 605 Lys Met Asn Ser Asp Ser Glu Glu Leu Thr Gln Arg Trp Asp Ser Leu 610 615 620 Val Gln Arg Leu Glu Asp Ser Ser Asn Gln Val Thr Gln Ala Val Ala 625 630 635 640 Lys Leu Gly Met Ser Gln Ile Pro Gln Lys Asp Leu Leu Glu Thr Val 645 650 655 His Val Arg Glu Lys Gly Met Val Lys Lys Pro Lys Gln Glu Leu Pro 660 665 670 Pro Pro Leu Thr Lys Ala Glu His Ala Met Gln Lys Arg Ser Thr Thr 675 680 685 Glu Leu Gly Glu Asn Leu Gln Glu Leu Arg Asp Leu Thr Gln Glu Met 690 695 700 Glu Val His Ala Glu Lys Leu Lys Trp Leu Asn Arg Thr Glu Leu Glu 705 710 715 720 Met Leu Ser Asp Lys Ser Leu Ser Leu Pro Glu Arg Asp Lys Ile Ser 725 730 735 Glu Ser Leu Arg Thr Val Asn Met Thr Trp Asn Lys Ile Cys Arg Glu 740 745 750 Val Pro Thr Thr Leu Lys Glu Cys Ile Gln Glu Pro Ser Ser Val Ser 755 760 765 Gln Thr Arg Ile Ala Ala His Pro Asn Val Gln Lys Val Val Leu Val 770 775 780 Ser Ser Ala Ser Asp Ile Pro Val Gln Ser His Arg Thr Ser Glu Ile 785 790 795 800 Ser Ile Pro Ala Asp Leu Asp Lys Thr Ile Thr Glu Leu Ala Asp Trp 805 810 815 Leu Val Leu Ile Asp Gln Met Leu Lys Ser Asn Ile Val Thr Val Gly 820 825 830 Asp Val Glu Glu Ile Asn Lys Thr Val Ser Arg Met Lys Ile Thr Lys 835 840 845 Ala Asp Leu Glu Gln Arg His Pro Gln Leu Asp Tyr Val Phe Thr Leu 850 855 860 Ala Gln Asn Leu Lys Asn Lys Ala Ser Ser Ser Asp Met Arg Thr Ala 865 870 875 880 Ile Thr Glu Lys Leu Glu Arg Val Lys Asn Gln Trp Asp Gly Thr Gln 885 890 895 His Gly Val Glu Leu Arg Gln Gln Gln Leu Glu Asp Met Ile Ile Asp 900 905 910 Ser Leu Gln Trp Asp Asp His Arg Glu Glu Thr Glu Glu Leu Met Arg 915 920 925 Lys Tyr Glu Ala Arg Leu Tyr Ile Leu Gln Gln Ala Arg Arg Asp Pro 930 935 940 Leu Thr Lys Gln Ile Ser Asp Asn Gln Ile Leu Leu Gln Glu Leu Gly 945 950 955 960 Pro Gly Asp Gly Ile Val Met Ala Phe Asp Asn Val Leu Gln Lys Leu 965 970 975 Leu Glu Glu Tyr Gly Ser Asp Asp Thr Arg Asn Val Lys Glu Thr Thr 980 985 990 Glu Tyr Leu Lys Thr Ser Trp Ile Asn Leu Lys Gln Ser Ile Ala Asp 995 1000 1005 Arg Gln Asn Ala Leu Glu Ala Glu Trp Arg Thr Val Gln Ala Ser Arg 1010 1015 1020 Arg Asp Leu Glu Asn Phe Leu Lys Trp Ile Gln Glu Ala Glu Thr Thr 1025 1030 1035 1040 Val Asn Val Leu Val Asp Ala Ser His Arg Glu Asn Ala Leu Gln Asp 1045 1050 1055 Ser Ile Leu Ala Arg Glu Leu Lys Gln Gln Met Gln Asp Ile Gln Ala 1060 1065 1070 Glu Ile Asp Ala His Asn Asp Ile Phe Lys Ser Ile Asp Gly Asn Arg 1075 1080 1085 Gln Lys Met Val Lys Ala Leu Gly Asn Ser Glu Glu Ala Thr Met Leu 1090 1095 1100 Gln His Arg Leu Asp Asp Met Asn Gln Arg Trp Asn Asp Leu Lys Ala 1105 1110 1115 1120 Lys Ser Ala Ser Ile Arg Ala His Leu Glu Ala Ser Ala Glu Lys Trp 1125 1130 1135 Asn Arg Leu Leu Met Ser Leu Glu Glu Leu Ile Lys Trp Leu Asn Met 1140 1145 1150 Lys Asp Glu Glu Leu Lys Lys Gln Met Pro Ile Gly Gly Asp Val Pro 1155 1160 1165 Ala Leu Gln Leu Gln Tyr Asp His Cys Lys Ala Leu Arg Arg Glu Leu 1170 1175 1180 Lys Glu Lys Glu Tyr Ser Val Leu Asn Ala Val Asp Gln Ala Arg Val 1185 1190 1195 1200 Phe Leu Ala Asp Gln Pro Ile Glu Ala Pro Glu Glu Pro Arg Arg Asn 1205 1210 1215 Leu Gln Ser Lys Thr Glu Leu Thr Pro Glu Glu Arg Ala Gln Lys Ile 1220 1225 1230 Ala Lys Ala Met Arg Lys Gln Ser Ser Glu Val Lys Glu Lys Trp Glu 1235 1240 1245 Ser Leu Asn Ala Val Thr Ser Asn Trp Gln Lys Gln Val Asp Lys Ala 1250 1255 1260 Leu Glu Lys Leu Arg Asp Leu Gln Gly Ala Met Asp Asp Leu Asp Ala 1265 1270 1275 1280 Asp Met Lys Glu Ala Glu Ser Val Arg Asn Gly Trp Lys Pro Val Gly 1285 1290 1295 Asp Leu Leu Ile Asp Ser Leu Gln Asp His Ile Glu Lys Ile Met Ala 1300 1305 1310 Phe Arg Glu Glu Ile Ala Pro Ile Asn Phe Lys Val Lys Thr Val Asn 1315 1320 1325 Asp Leu Ser Ser Gln Leu Ser Pro Leu Asp Leu His Pro Ser Leu Lys 1330 1335 1340 Met Ser Arg Gln Leu Asp Asp Leu Asn Met Arg Trp Lys Leu Leu Gln 1345 1350 1355 1360 Val Ser Val Asp Asp Arg Leu Lys Gln Leu Gln Glu Ala His Arg Asp 1365 1370 1375 Phe Gly Pro Ser Ser Gln His Phe Leu Ser Thr Ser Val Gln Leu Pro 1380 1385 1390 Trp Gln Arg Ser Ile Ser His Asn Lys Val Pro Tyr Tyr Ile Asn His 1395 1400 1405 Gln Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu Phe 1410 1415 1420 Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr 1425 1430 1435 1440 Ala Ile Lys Ile Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu 1445 1450 1455 Glu Leu Ser Thr Thr Asn Glu Ile Phe Lys Gln His Lys Leu Asn Gln 1460 1465 1470 Asn Asp Gln Leu Leu Ser Val Pro Asp Val Ile Asn Cys Leu Thr Thr 1475 1480 1485 Thr Tyr Asp Gly Leu Glu Gln Met His Lys Asp Leu Val Asn Val Pro 1490 1495 1500 Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr 1505 1510 1515 1520 Gly Arg Thr Gly Lys Ile Arg Val Gln Ser Leu Lys Ile Gly Leu Met 1525 1530 1535 Ser Leu Ser Lys Gly Leu Leu Glu Glu Lys Tyr Arg Tyr Leu Phe Lys 1540 1545 1550 Glu Val Ala Gly Pro Thr Glu Met Cys Asp Gln Arg Gln Leu Gly Leu 1555 1560 1565 Leu Leu His Asp Ala Ile Gln Ile Pro Arg Gln Leu Gly Glu Val Ala 1570 1575 1580 Ala Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser Cys Phe Gln 1585 1590 1595 1600 Gln Asn Asn Asn Lys Pro Glu Ile Ser Val Lys Glu Phe Ile Asp Trp 1605 1610 1615 Met His Leu Glu Pro Gln Ser Met Val Trp Leu Pro Val Leu His Arg 1620 1625 1630 Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys 1635 1640 1645 Lys Glu Cys Pro Ile Val Gly Phe Arg Tyr Arg Ser Leu Lys His Phe 1650 1655 1660 Asn Tyr Asp Val Cys Gln Ser Cys Phe Phe Ser Gly Arg Thr Ala Lys 1665 1670 1675 1680 Gly His Lys Leu His Tyr Pro Met Val Glu Tyr Cys Ile Pro Thr Thr 1685 1690 1695 Ser Gly Glu Asp Val Arg Asp Phe Thr Lys Val Leu Lys Asn Lys Phe 1700 1705 1710 Arg Ser Lys Lys Tyr Phe Ala Lys His Pro Arg Leu Gly Tyr Leu Pro 1715 1720 1725 Val Gln Thr Val Leu Glu Gly Asp Asn Leu Glu Thr Pro Ile Thr Leu 1730 1735 1740 Ile Ser Met Trp Pro Glu His Tyr Asp Pro Ser Gln Ser Pro Gln Leu 1745 1750 1755 1760 Phe His Asp Asp Thr His Ser Arg Ile Glu Gln Tyr Ala Thr Arg Leu 1765 1770 1775 Ala Gln Met Glu Arg Thr Asn Gly Ser Phe Leu Thr Asp Ser Ser Ser 1780 1785 1790 Thr Thr Gly Ser Val Glu Asp Glu His Ala Leu Ile Gln Gln Tyr Cys 1795 1800 1805 Gln Thr Leu Gly Gly Glu Ser Pro Val Ser Gln Pro Gln Ser Pro Ala 1810 1815 1820 Gln Ile Leu Lys Ser Val Glu Arg Glu Glu Arg Gly Glu Leu Glu Arg 1825 1830 1835 1840 Ile Ile Ala Asp Leu Glu Glu Glu Gln Arg Asn Leu Gln Val Glu Tyr 1845 1850 1855 Glu Gln Leu Lys Asp Gln His Leu Arg Arg Gly Leu Pro Val Gly Ser 1860 1865 1870 Pro Pro Glu Ser Ile Ile Ser Pro His His Thr Ser Glu Asp Ser Glu 1875 1880 1885 Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu Glu 1890 1895 1900 Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu Glu Ser Gln 1905 1910 1915 1920 Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Glu Ser Asp Ser Arg 1925 1930 1935 Ile Asn Gly Val Ser Pro Trp Ala Ser Pro Gln His Ser Ala Leu Ser 1940 1945 1950 Tyr Ser Leu Asp Pro Asp Ala Ser Gly Pro Gln Phe His Gln Ala Ala 1955 1960 1965 Gly Glu Asp Leu Leu Ala Pro Pro His Asp Thr Ser Thr Asp Leu Thr 1970 1975 1980 Glu Val Met Glu Gln Ile His Ser Thr Phe Pro Ser Cys Cys Pro Asn 1985 1990 1995 2000 Val Pro Ser Arg Pro Gln Ala Met 2005 9 10320 DNA Artificial Sequence CDS (11)..(10312) Description of Artificial Sequence Full length utrophin construct 9 actagtcaag atg gcc aag tat gga gaa cat gaa gcc agt cct gac aat 49 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn 1 5 10 ggg cag aac gaa ttc agt gac atc att gag tcc aga tct gat gaa cac 97 Gly Gln Asn Glu Phe Ser Asp Ile Ile Glu Ser Arg Ser Asp Glu His 15 20 25 aat gat gta cag aag aaa acc ttt acc aaa tgg ata aac gct cga ttt 145 Asn Asp Val Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe 30 35 40 45 tcc aag agt ggg aaa cca ccc atc agt gat atg ttc tca gac ctc aaa 193 Ser Lys Ser Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys 50 55 60 gat ggg aga aag ctc ttg gat ctt ctc gaa ggc ctc aca gga aca tca 241 Asp Gly Arg Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser 65 70 75 ttg cca aag gaa cgt ggt tcc aca agg gtg cat gcc tta aac aat gtc 289 Leu Pro Lys Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val 80 85 90 aac cga gtg cta cag gtt tta cat cag aac aat gtg gac ttg gtg aat 337 Asn Arg Val Leu Gln Val Leu His Gln Asn Asn Val Asp Leu Val Asn 95 100 105 att gga ggc acg gac att gtg gat gga aat ccc aag ctg act tta ggg 385 Ile Gly Gly Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly 110 115 120 125 tta ctc tgg agc atc att ctg cac tgg cag gtg aag gat gtc atg aaa 433 Leu Leu Trp Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys 130 135 140 gat atc atg tca gac ctg cag cag aca aac agc gag aag atc ctg ctg 481 Asp Ile Met Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu 145 150 155 agc tgg gtg cgg cag acc acc agg ccc tac agt caa gtc aac gtc ctc 529 Ser Trp Val Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu 160 165 170 aac ttc acc acc agc tgg acc gat gga ctc gcg ttc aac gcc gtg ctc 577 Asn Phe Thr Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu 175 180 185 cac cgg cac aaa cca gat ctc ttc agc tgg gac aga gtg gtc aaa atg 625 His Arg His Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met 190 195 200 205 tcc cca att gag aga ctt gaa cat gct ttt agc aag gcc cac act tat 673 Ser Pro Ile Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr 210 215 220 ttg gga att gaa aag ctt cta gat cct gaa gat gtt gct gtg cat ctc 721 Leu Gly Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val His Leu 225 230 235 ccn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn ncc gtt gag gtg 769 Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Glu Val 240 245 250 ctt cct cag caa gtc acg ata gat gcc atc cga gag gtg gag act ctc 817 Leu Pro Gln Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu 255 260 265 cca agg aag tat aag aaa gaa tgt gaa gag gaa gaa att cat atc cag 865 Pro Arg Lys Tyr Lys Lys Glu Cys Glu Glu Glu Glu Ile His Ile Gln 270 275 280 285 agt gca gtg ctg gca gag gaa ggc cag agt ccc cga gct gag acc cct 913 Ser Ala Val Leu Ala Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr Pro 290 295 300 agc acc gtc act gaa gtg gac atg gat ttg gac agc tac cag ata gcg 961 Ser Thr Val Thr Glu Val Asp Met Asp Leu Asp Ser Tyr Gln Ile Ala 305 310 315 cta gag gaa gtg ctg acg tgg ctg ctg tcc gcg gag gac acg ttc cag 1009 Leu Glu Glu Val Leu Thr Trp Leu Leu Ser Ala Glu Asp Thr Phe Gln 320 325 330 gag caa gat gac att tct gat gat gtc gaa gaa gtc aaa gag cag ttt 1057 Glu Gln Asp Asp Ile Ser Asp Asp Val Glu Glu Val Lys Glu Gln Phe 335 340 345 gct acc cat gaa act ttt atg atg gag ctg aca gca cac cag agc agc 1105 Ala Thr His Glu Thr Phe Met Met Glu Leu Thr Ala His Gln Ser Ser 350 355 360 365 gtg ggg agc gtc ctg cag gct ggc aac cag ctg atg aca caa ggg act 1153 Val Gly Ser Val Leu Gln Ala Gly Asn Gln Leu Met Thr Gln Gly Thr 370 375 380 ctg tca gag gag gag gag ttt gag atc cag gaa cag atg acc ttg ctg 1201 Leu Ser Glu Glu Glu Glu Phe Glu Ile Gln Glu Gln Met Thr Leu Leu 385 390 395 aat gca agg tgg gag gcg ctc cgg gtg gag agc atg gag agg cag tcc 1249 Asn Ala Arg Trp Glu Ala Leu Arg Val Glu Ser Met Glu Arg Gln Ser 400 405 410 cgg ctg cac gac gct ctg atg gag ctg cag aag aaa cag ctg cag cag 1297 Arg Leu His Asp Ala Leu Met Glu Leu Gln Lys Lys Gln Leu Gln Gln 415 420 425 ctc tca agc tgg ctg gcc ctc aca gaa gag cgc att cag aag atg gag 1345 Leu Ser Ser Trp Leu Ala Leu Thr Glu Glu Arg Ile Gln Lys Met Glu 430 435 440 445 agc cct ccg ctg ggt gat gac ctg ccc tcc ctg cag aag ctg ctt caa 1393 Ser Pro Pro Leu Gly Asp Asp Leu Pro Ser Leu Gln Lys Leu Leu Gln 450 455 460 gaa cat aaa agt ttg caa aat gac ctt gaa gct gaa cag gtg aag gta 1441 Glu His Lys Ser Leu Gln Asn Asp Leu Glu Ala Glu Gln Val Lys Val 465 470 475 aat tcc tta act cac atg gtg gtg att gtg gat gaa aac agt ggg gag 1489 Asn Ser Leu Thr His Met Val Val Ile Val Asp Glu Asn Ser Gly Glu 480 485 490 agt gcc aca gct ctt ctg gaa gat cag tta cag aaa ctg ggt gag cgc 1537 Ser Ala Thr Ala Leu Leu Glu Asp Gln Leu Gln Lys Leu Gly Glu Arg 495 500 505 tgg aca gct gta tgc cgc tgg act gaa gaa cgt tgg aac agg ttg caa 1585 Trp Thr Ala Val Cys Arg Trp Thr Glu Glu Arg Trp Asn Arg Leu Gln 510 515 520 525 gaa atc agt att ctg tgg cag gaa tta ttg gaa gag cag tgt ctg ttg 1633 Glu Ile Ser Ile Leu Trp Gln Glu Leu Leu Glu Glu Gln Cys Leu Leu 530 535 540 gag gct tgg ctc acc gaa aag gaa gag gct ttg aat aaa gtt caa acc 1681 Glu Ala Trp Leu Thr Glu Lys Glu Glu Ala Leu Asn Lys Val Gln Thr 545 550 555 agc aac ttt aaa gac cag aag gaa cta agt gtc agt gtc cgg cgt ctg 1729 Ser Asn Phe Lys Asp Gln Lys Glu Leu Ser Val Ser Val Arg Arg Leu 560 565 570 gct ata ttg aag gaa gac atg gaa atg aag agg cag act ctg gat caa 1777 Ala Ile Leu Lys Glu Asp Met Glu Met Lys Arg Gln Thr Leu Asp Gln 575 580 585 ctg agt gag att ggc cag gat gtg ggc caa tta ctc agt aat ccc aag 1825 Leu Ser Glu Ile Gly Gln Asp Val Gly Gln Leu Leu Ser Asn Pro Lys 590 595 600 605 gca tct aag aag atg aac agt gac tct gag gag cta aca cag aga tgg 1873 Ala Ser Lys Lys Met Asn Ser Asp Ser Glu Glu Leu Thr Gln Arg Trp 610 615 620 gat tct ctg gtt cag aga ctc gaa gac tct tct aac cag gtg act cag 1921 Asp Ser Leu Val Gln Arg Leu Glu Asp Ser Ser Asn Gln Val Thr Gln 625 630 635 gcg gta gcg aag ctc ggc atg tcc cag att cca cag aag gac cta ttg 1969 Ala Val Ala Lys Leu Gly Met Ser Gln Ile Pro Gln Lys Asp Leu Leu 640 645 650 gag acc gtt cat gtg aga gaa aaa ggg atg gtg aag aag ccc aag cag 2017 Glu Thr Val His Val Arg Glu Lys Gly Met Val Lys Lys Pro Lys Gln 655 660 665 gaa ctg cct cct ccg ttg ggc cca aag aag aga cag atc cat gtg gat 2065 Glu Leu Pro Pro Pro Leu Gly Pro Lys Lys Arg Gln Ile His Val Asp 670 675 680 685 att gaa gct aag aaa aag ttt gat gct ata agt gca gag ctg ttg aac 2113 Ile Glu Ala Lys Lys Lys Phe Asp Ala Ile Ser Ala Glu Leu Leu Asn 690 695 700 tgg att ttg aaa tgg aaa act gcc att cag acc aca gag ata aaa gag 2161 Trp Ile Leu Lys Trp Lys Thr Ala Ile Gln Thr Thr Glu Ile Lys Glu 705 710 715 tat atg aag atg caa gac act tcc gaa atg aaa aag aag ttg aag gca 2209 Tyr Met Lys Met Gln Asp Thr Ser Glu Met Lys Lys Lys Leu Lys Ala 720 725 730 tta gaa aaa gaa cag aga gaa aga atc ccc aga gca gat gaa tta aac 2257 Leu Glu Lys Glu Gln Arg Glu Arg Ile Pro Arg Ala Asp Glu Leu Asn 735 740 745 caa act gga caa atc ctt gtg gag caa atg gga aaa gaa ggc ctt cct 2305 Gln Thr Gly Gln Ile Leu Val Glu Gln Met Gly Lys Glu Gly Leu Pro 750 755 760 765 act gaa gaa ata aaa aat gtt ctg gag aag gtt tca tca gaa tgg aag 2353 Thr Glu Glu Ile Lys Asn Val Leu Glu Lys Val Ser Ser Glu Trp Lys 770 775 780 aat gta tct caa cat ttg gaa gat cta gaa aga aag att cag cta cag 2401 Asn Val Ser Gln His Leu Glu Asp Leu Glu Arg Lys Ile Gln Leu Gln 785 790 795 gaa gat ata aat gct tat ttc aag cag ctt gat gag ctt gaa aag gtc 2449 Glu Asp Ile Asn Ala Tyr Phe Lys Gln Leu Asp Glu Leu Glu Lys Val 800 805 810 atc aag aca aag gag gag tgg gta aaa cac act tcc att tct gaa tct 2497 Ile Lys Thr Lys Glu Glu Trp Val Lys His Thr Ser Ile Ser Glu Ser 815 820 825 tcc cgg cag tcc ttg cca agc ttg aag gat tcc tgt cag cgg gaa ttg 2545 Ser Arg Gln Ser Leu Pro Ser Leu Lys Asp Ser Cys Gln Arg Glu Leu 830 835 840 845 aca aat ctt ctt ggc ctt cac ccc aaa att gaa atg gct cgt gca agc 2593 Thr Asn Leu Leu Gly Leu His Pro Lys Ile Glu Met Ala Arg Ala Ser 850 855 860 tgc tcg gcc ctg atg tct cag cct tct gcc cca gat ttt gtc cag cgg 2641 Cys Ser Ala Leu Met Ser Gln Pro Ser Ala Pro Asp Phe Val Gln Arg 865 870 875 ggc ttc gat agc ttt ctg ggc cgc tac caa gct gta caa gag gct gta 2689 Gly Phe Asp Ser Phe Leu Gly Arg Tyr Gln Ala Val Gln Glu Ala Val 880 885 890 gag gat cgt caa caa cat cta gag aat gaa ctg aag ggc caa cct gga 2737 Glu Asp Arg Gln Gln His Leu Glu Asn Glu Leu Lys Gly Gln Pro Gly 895 900 905 cat gca tat ctg gaa aca ttg aaa aca ctg aaa gat gtg cta aat gat 2785 His Ala Tyr Leu Glu Thr Leu Lys Thr Leu Lys Asp Val Leu Asn Asp 910 915 920 925 tca gaa aat aag gcc cag gtg tct ctg aat gtc ctt aat gat ctt gcc 2833 Ser Glu Asn Lys Ala Gln Val Ser Leu Asn Val Leu Asn Asp Leu Ala 930 935 940 aag gtg gag aag gcc ctg caa gaa aaa aag acc ctt gat gaa atc ctt 2881 Lys Val Glu Lys Ala Leu Gln Glu Lys Lys Thr Leu Asp Glu Ile Leu 945 950 955 gag aat cag aaa cct gca tta cat aaa ctt gca gaa gaa aca aag gct 2929 Glu Asn Gln Lys Pro Ala Leu His Lys Leu Ala Glu Glu Thr Lys Ala 960 965 970 ctg gag aaa aat gtt cat cct gat gta gaa aaa tta tat aag caa gaa 2977 Leu Glu Lys Asn Val His Pro Asp Val Glu Lys Leu Tyr Lys Gln Glu 975 980 985 ttt gat gat gtg caa gga aag tgg aac aag cta aag gtc ttg gtt tcc 3025 Phe Asp Asp Val Gln Gly Lys Trp Asn Lys Leu Lys Val Leu Val Ser 990 995 1000 1005 aaa gat cta cat ttg ctt gag gaa att gct ctc aca ctc aga gct ttt 3073 Lys Asp Leu His Leu Leu Glu Glu Ile Ala Leu Thr Leu Arg Ala Phe 1010 1015 1020 gag gcc gat tca aca gtc att gag aag tgg atg gat ggc gtg aaa gac 3121 Glu Ala Asp Ser Thr Val Ile Glu Lys Trp Met Asp Gly Val Lys Asp 1025 1030 1035 ttc tta atg aaa cag cag gct gcc caa gga gac gac gca ggt cta cag 3169 Phe Leu Met Lys Gln Gln Ala Ala Gln Gly Asp Asp Ala Gly Leu Gln 1040 1045 1050 agg cag tta gac cag tgc tct gca ttt gtt aat gaa ata gaa aca att 3217 Arg Gln Leu Asp Gln Cys Ser Ala Phe Val Asn Glu Ile Glu Thr Ile 1055 1060 1065 gaa tca tct ctg aaa aac atg aag gaa ata gag act aat ctt cga agt 3265 Glu Ser Ser Leu Lys Asn Met Lys Glu Ile Glu Thr Asn Leu Arg Ser 1070 1075 1080 1085 ggt cca gtt gct gga ata aaa act tgg gtg cag aca aga cta ggt gac 3313 Gly Pro Val Ala Gly Ile Lys Thr Trp Val Gln Thr Arg Leu Gly Asp 1090 1095 1100 tac caa act caa ctg gag aaa ctt agc aag gag atc gct act caa aaa 3361 Tyr Gln Thr Gln Leu Glu Lys Leu Ser Lys Glu Ile Ala Thr Gln Lys 1105 1110 1115 agt agg ttg tct gaa agt caa gaa aaa gct gcg aac ctg aag aaa gac 3409 Ser Arg Leu Ser Glu Ser Gln Glu Lys Ala Ala Asn Leu Lys Lys Asp 1120 1125 1130 ttg gca gag atg cag gaa tgg atg acc cag gcc gag gaa gaa tat ttg 3457 Leu Ala Glu Met Gln Glu Trp Met Thr Gln Ala Glu Glu Glu Tyr Leu 1135 1140 1145 gag cgg gat ttt gag tac aag tca cca gaa gag ctt gag agt gct gtg 3505 Glu Arg Asp Phe Glu Tyr Lys Ser Pro Glu Glu Leu Glu Ser Ala Val 1150 1155 1160 1165 gaa gag atg aag agg gca aaa gag gat gtg ttg cag aag gag gtg aga 3553 Glu Glu Met Lys Arg Ala Lys Glu Asp Val Leu Gln Lys Glu Val Arg 1170 1175 1180 gtg aag att ctc aag gac aac atc aag tta tta gct gcc aag gtg ccc 3601 Val Lys Ile Leu Lys Asp Asn Ile Lys Leu Leu Ala Ala Lys Val Pro 1185 1190 1195 tct ggt ggc cag gag ttg acg tct gag ctg aat gtt gtg ctg gag aat 3649 Ser Gly Gly Gln Glu Leu Thr Ser Glu Leu Asn Val Val Leu Glu Asn 1200 1205 1210 tac caa ctt ctt tgt aat aga att cga gga aag tgc cac acg cta gag 3697 Tyr Gln Leu Leu Cys Asn Arg Ile Arg Gly Lys Cys His Thr Leu Glu 1215 1220 1225 gag gtc tgg tct tgt tgg att gaa ctg ctt cac tat ttg gat ctt gaa 3745 Glu Val Trp Ser Cys Trp Ile Glu Leu Leu His Tyr Leu Asp Leu Glu 1230 1235 1240 1245 act acc tgg tta aac act ttg gaa gag cgg atg aag agc aca gag gtc 3793 Thr Thr Trp Leu Asn Thr Leu Glu Glu Arg Met Lys Ser Thr Glu Val 1250 1255 1260 ctg cct gag aag acg gat gct gtc aac gaa gcc ctg gag tct ctg gaa 3841 Leu Pro Glu Lys Thr Asp Ala Val Asn Glu Ala Leu Glu Ser Leu Glu 1265 1270 1275 tct gtt ctg cgc cac ccg gca gat aat cgc acc cag att cga gag ctt 3889 Ser Val Leu Arg His Pro Ala Asp Asn Arg Thr Gln Ile Arg Glu Leu 1280 1285 1290 ggc cag act ctg att gat ggg ggg atc ctg gat gat ata atc agt gag 3937 Gly Gln Thr Leu Ile Asp Gly Gly Ile Leu Asp Asp Ile Ile Ser Glu 1295 1300 1305 aaa ctg gag gct ttc aac agc cga tat gaa gat cta agt cac ctg gca 3985 Lys Leu Glu Ala Phe Asn Ser Arg Tyr Glu Asp Leu Ser His Leu Ala 1310 1315 1320 1325 gag agc aag cag att tct ttg gaa aag caa ctc cag gtg ctg cgg gaa 4033 Glu Ser Lys Gln Ile Ser Leu Glu Lys Gln Leu Gln Val Leu Arg Glu 1330 1335 1340 act gac cag atg ctt caa gtc ttg caa gag agc ttg ggg gag ctg gac 4081 Thr Asp Gln Met Leu Gln Val Leu Gln Glu Ser Leu Gly Glu Leu Asp 1345 1350 1355 aaa cag ctc acc aca tac ctg act gac agg ata gat gct ttc caa gtt 4129 Lys Gln Leu Thr Thr Tyr Leu Thr Asp Arg Ile Asp Ala Phe Gln Val 1360 1365 1370 cca cag gaa gct cag aaa atc caa gca gag atc tca gcc cat gag cta 4177 Pro Gln Glu Ala Gln Lys Ile Gln Ala Glu Ile Ser Ala His Glu Leu 1375 1380 1385 acc cta gag gag ttg aga aga aat atg cgt tct cag ccc ctg acc tcc 4225 Thr Leu Glu Glu Leu Arg Arg Asn Met Arg Ser Gln Pro Leu Thr Ser 1390 1395 1400 1405 cca gag agt agg act gcc aga gga gga agt cag atg gat gtg cta cag 4273 Pro Glu Ser Arg Thr Ala Arg Gly Gly Ser Gln Met Asp Val Leu Gln 1410 1415 1420 agg aaa ctc cga gag gtg tcc aca aag ttc cag ctt ttc cag aag cca 4321 Arg Lys Leu Arg Glu Val Ser Thr Lys Phe Gln Leu Phe Gln Lys Pro 1425 1430 1435 gct aac ttc gag cag cgc atg ctg gac tgc aag cgt gtg ctg gat ggc 4369 Ala Asn Phe Glu Gln Arg Met Leu Asp Cys Lys Arg Val Leu Asp Gly 1440 1445 1450 gtg aaa gca gaa ctt cac gtt ctg gat gtg aag gac gta gac cct gac 4417 Val Lys Ala Glu Leu His Val Leu Asp Val Lys Asp Val Asp Pro Asp 1455 1460 1465 gtc ata cag acg cac ctg gac aag tgt atg aaa ctg tat aaa act ttg 4465 Val Ile Gln Thr His Leu Asp Lys Cys Met Lys Leu Tyr Lys Thr Leu 1470 1475 1480 1485 agt gaa gtc aaa ctt gaa gtg gaa act gtg att aaa aca gga aga cat 4513 Ser Glu Val Lys Leu Glu Val Glu Thr Val Ile Lys Thr Gly Arg His 1490 1495 1500 att gtc cag aaa cag caa acg gac aac cca aaa ggg atg gat gag cag 4561 Ile Val Gln Lys Gln Gln Thr Asp Asn Pro Lys Gly Met Asp Glu Gln 1505 1510 1515 ctg act tcc ctg aag gtt ctt tac aat gac ctg ggc gca cag gtg aca 4609 Leu Thr Ser Leu Lys Val Leu Tyr Asn Asp Leu Gly Ala Gln Val Thr 1520 1525 1530 gaa gga aaa cag gat ctg gaa aga gca tca cag ttg gcc cgg aaa atg 4657 Glu Gly Lys Gln Asp Leu Glu Arg Ala Ser Gln Leu Ala Arg Lys Met 1535 1540 1545 aag aaa gag gct gct tct ctc tct gaa tgg ctt tct gct act gaa act 4705 Lys Lys Glu Ala Ala Ser Leu Ser Glu Trp Leu Ser Ala Thr Glu Thr 1550 1555 1560 1565 gaa ttg gta cag aag tcc act tca gaa ggt ctg ctt ggt gac ttg gat 4753 Glu Leu Val Gln Lys Ser Thr Ser Glu Gly Leu Leu Gly Asp Leu Asp 1570 1575 1580 aca gaa att tcc tgg gct aaa aat gtt ctg aag gat ctg gaa aag aga 4801 Thr Glu Ile Ser Trp Ala Lys Asn Val Leu Lys Asp Leu Glu Lys Arg 1585 1590 1595 aaa gct gat tta aat acc atc aca gag agt agt gct gcc ctg caa aac 4849 Lys Ala Asp Leu Asn Thr Ile Thr Glu Ser Ser Ala Ala Leu Gln Asn 1600 1605 1610 ttg att gag ggc agt gag cct att tta gaa gag agg ctc tgc gtc ctt 4897 Leu Ile Glu Gly Ser Glu Pro Ile Leu Glu Glu Arg Leu Cys Val Leu 1615 1620 1625 aac gct ggg tgg agc cga gtt cgt acc tgg act gaa gat tgg tgc aat 4945 Asn Ala Gly Trp Ser Arg Val Arg Thr Trp Thr Glu Asp Trp Cys Asn 1630 1635 1640 1645 acc ttg atg aac cat cag aac cag cta gaa ata ttt gat ggg aac gtg 4993 Thr Leu Met Asn His Gln Asn Gln Leu Glu Ile Phe Asp Gly Asn Val 1650 1655 1660 gct cac ata agt acc tgg ctt tat caa gct gaa gct cta ttg gat gaa 5041 Ala His Ile Ser Thr Trp Leu Tyr Gln Ala Glu Ala Leu Leu Asp Glu 1665 1670 1675 att gaa aag aaa cca aca agt aaa cag gaa gaa att gtg aag cgt tta 5089 Ile Glu Lys Lys Pro Thr Ser Lys Gln Glu Glu Ile Val Lys Arg Leu 1680 1685 1690 gta tct gag ctg gat gat gcc aac ctc cag gtt gaa aat gtc cgc gat 5137 Val Ser Glu Leu Asp Asp Ala Asn Leu Gln Val Glu Asn Val Arg Asp 1695 1700 1705 caa gcc ctt att ttg atg aat gcc cgt gga agc tca agc agg gag ctt 5185 Gln Ala Leu Ile Leu Met Asn Ala Arg Gly Ser Ser Ser Arg Glu Leu 1710 1715 1720 1725 gta gaa cca aag tta gct gag ctg aat agg aac ttt gaa aag gtg tct 5233 Val Glu Pro Lys Leu Ala Glu Leu Asn Arg Asn Phe Glu Lys Val Ser 1730 1735 1740 caa cat atc aaa agt gcc aaa ttg cta att gct cag gaa cca tta tac 5281 Gln His Ile Lys Ser Ala Lys Leu Leu Ile Ala Gln Glu Pro Leu Tyr 1745 1750 1755 caa tgt ttg gtc acc act gaa aca ttt gaa act ggt gtg cct ttc tct 5329 Gln Cys Leu Val Thr Thr Glu Thr Phe Glu Thr Gly Val Pro Phe Ser 1760 1765 1770 gac ttg gaa aaa tta gaa aat gac ata gaa aat atg tta aaa ttt gtg 5377 Asp Leu Glu Lys Leu Glu Asn Asp Ile Glu Asn Met Leu Lys Phe Val 1775 1780 1785 gaa aaa cac ttg gaa tcc agt gat gaa gat gaa aag atg gat gag gag 5425 Glu Lys His Leu Glu Ser Ser Asp Glu Asp Glu Lys Met Asp Glu Glu 1790 1795 1800 1805 agt gcc cag att gag gaa gtt cta caa aga gga gaa gaa atg tta cat 5473 Ser Ala Gln Ile Glu Glu Val Leu Gln Arg Gly Glu Glu Met Leu His 1810 1815 1820 caa cct atg gaa gat aat aaa aaa gaa aag atc cgt ttg caa tta tta 5521 Gln Pro Met Glu Asp Asn Lys Lys Glu Lys Ile Arg Leu Gln Leu Leu 1825 1830 1835 ctt ttg cat act aga tac aac aaa att aag gca atc cct att caa cag 5569 Leu Leu His Thr Arg Tyr Asn Lys Ile Lys Ala Ile Pro Ile Gln Gln 1840 1845 1850 agg aaa atg ggt caa ctt gct tct gga att aga tca tca ctt ctt cct 5617 Arg Lys Met Gly Gln Leu Ala Ser Gly Ile Arg Ser Ser Leu Leu Pro 1855 1860 1865 aca gat tat ctg gtt gaa att aac aaa att tta ctt tgc atg gat gat 5665 Thr Asp Tyr Leu Val Glu Ile Asn Lys Ile Leu Leu Cys Met Asp Asp 1870 1875 1880 1885 gtt gaa tta tcg ctt aat gtt cca gag ctc aac act gct att tac gaa 5713 Val Glu Leu Ser Leu Asn Val Pro Glu Leu Asn Thr Ala Ile Tyr Glu 1890 1895 1900 gac ttc tct ttt cag gaa gac tct ctg aag aat atc aaa gac caa ctg 5761 Asp Phe Ser Phe Gln Glu Asp Ser Leu Lys Asn Ile Lys Asp Gln Leu 1905 1910 1915 gac aaa ctt gga gag cag att gca gtc att cat gaa aaa cag cca gat 5809 Asp Lys Leu Gly Glu Gln Ile Ala Val Ile His Glu Lys Gln Pro Asp 1920 1925 1930 gtc atc ctt gaa gcc tct gga cct gaa gcc att cag atc aga gat aca 5857 Val Ile Leu Glu Ala Ser Gly Pro Glu Ala Ile Gln Ile Arg Asp Thr 1935 1940 1945 ctt act cag ctg aat gca aaa tgg gac aga att aat aga atg tac agt 5905 Leu Thr Gln Leu Asn Ala Lys Trp Asp Arg Ile Asn Arg Met Tyr Ser 1950 1955 1960 1965 gat cgg aaa ggt tgt ttt gac agg gca atg gaa gaa tgg aga cag ttc 5953 Asp Arg Lys Gly Cys Phe Asp Arg Ala Met Glu Glu Trp Arg Gln Phe 1970 1975 1980 cat tgt gac ctt aat gac ctc aca cag tgg ata aca gag gct gaa gaa 6001 His Cys Asp Leu Asn Asp Leu Thr Gln Trp Ile Thr Glu Ala Glu Glu 1985 1990 1995 tta ctg gtt gat acc tgt gct cca ggt ggc agc ctg gac tta gag aaa 6049 Leu Leu Val Asp Thr Cys Ala Pro Gly Gly Ser Leu Asp Leu Glu Lys 2000 2005 2010 gcc agg ata cat cag cag gaa ctt gag gtg ggc atc agc agc cac cag 6097 Ala Arg Ile His Gln Gln Glu Leu Glu Val Gly Ile Ser Ser His Gln 2015 2020 2025 ccc agt ttt gca gca cta aac cga act ggg gat ggg att gtg cag aaa 6145 Pro Ser Phe Ala Ala Leu Asn Arg Thr Gly Asp Gly Ile Val Gln Lys 2030 2035 2040 2045 ctc tcc cag gca gat gga agc ttc ttg aaa gaa aaa ctg gca ggt tta 6193 Leu Ser Gln Ala Asp Gly Ser Phe Leu Lys Glu Lys Leu Ala Gly Leu 2050 2055 2060 aac caa cgc tgg gat gca att gtt gca gaa gtg aag gat agg cag cca 6241 Asn Gln Arg Trp Asp Ala Ile Val Ala Glu Val Lys Asp Arg Gln Pro 2065 2070 2075 agg cta aaa gga gaa agt aag cag gtg atg aag tac agg cat cag cta 6289 Arg Leu Lys Gly Glu Ser Lys Gln Val Met Lys Tyr Arg His Gln Leu 2080 2085 2090 gat gag att atc tgt tgg tta aca aag gct gag cat gct atg caa aag 6337 Asp Glu Ile Ile Cys Trp Leu Thr Lys Ala Glu His Ala Met Gln Lys 2095 2100 2105 aga tca acc acc gaa ttg gga gaa aac ctg caa gaa tta aga gac tta 6385 Arg Ser Thr Thr Glu Leu Gly Glu Asn Leu Gln Glu Leu Arg Asp Leu 2110 2115 2120 2125 act caa gaa atg gaa gta cat gct gaa aaa ctc aaa tgg ctg aat aga 6433 Thr Gln Glu Met Glu Val His Ala Glu Lys Leu Lys Trp Leu Asn Arg 2130 2135 2140 act gaa ttg gag atg ctt tca gat aaa agt ctg agt tta cct gaa agg 6481 Thr Glu Leu Glu Met Leu Ser Asp Lys Ser Leu Ser Leu Pro Glu Arg 2145 2150 2155 gat aaa att tca gaa agc tta agg act gta aat atg aca tgg aat aag 6529 Asp Lys Ile Ser Glu Ser Leu Arg Thr Val Asn Met Thr Trp Asn Lys 2160 2165 2170 att tgc aga gag gtg cct acc acc ctg aag gaa tgc atc cag gag ccc 6577 Ile Cys Arg Glu Val Pro Thr Thr Leu Lys Glu Cys Ile Gln Glu Pro 2175 2180 2185 agt tct gtt tca cag aca agg att gct gct cat cct aat gtc caa aag 6625 Ser Ser Val Ser Gln Thr Arg Ile Ala Ala His Pro Asn Val Gln Lys 2190 2195 2200 2205 gtg gtg cta gta tca tct gcg tca gat att cct gtt cag tct cat cgt 6673 Val Val Leu Val Ser Ser Ala Ser Asp Ile Pro Val Gln Ser His Arg 2210 2215 2220 act tcg gaa att tca att cct gct gat ctt gat aaa act ata aca gaa 6721 Thr Ser Glu Ile Ser Ile Pro Ala Asp Leu Asp Lys Thr Ile Thr Glu 2225 2230 2235 cta gcc gac tgg ctg gta tta atc gac cag atg ctg aag tcc aac att 6769 Leu Ala Asp Trp Leu Val Leu Ile Asp Gln Met Leu Lys Ser Asn Ile 2240 2245 2250 gtc act gtt ggg gat gta gaa gag atc aat aag acc gtt tcc cga atg 6817 Val Thr Val Gly Asp Val Glu Glu Ile Asn Lys Thr Val Ser Arg Met 2255 2260 2265 aaa att aca aag gct gac tta gaa cag cgc cat cct cag ctg gat tat 6865 Lys Ile Thr Lys Ala Asp Leu Glu Gln Arg His Pro Gln Leu Asp Tyr 2270 2275 2280 2285 gtt ttt aca ttg gca cag aat ttg aaa aat aaa gct tcc agt tca gat 6913 Val Phe Thr Leu Ala Gln Asn Leu Lys Asn Lys Ala Ser Ser Ser Asp 2290 2295 2300 atg aga aca gca att aca gaa aaa ttg gaa agg gtc aag aac cag tgg 6961 Met Arg Thr Ala Ile Thr Glu Lys Leu Glu Arg Val Lys Asn Gln Trp 2305 2310 2315 gat ggc acc cag cat ggc gtt gag cta aga cag cag cag ctt gag gac 7009 Asp Gly Thr Gln His Gly Val Glu Leu Arg Gln Gln Gln Leu Glu Asp 2320 2325 2330 atg att att gac agt ctt cag tgg gat gac cat agg gag gag act gaa 7057 Met Ile Ile Asp Ser Leu Gln Trp Asp Asp His Arg Glu Glu Thr Glu 2335 2340 2345 gaa ctg atg aga aaa tat gag gct cga ctc tat att ctt cag caa gcc 7105 Glu Leu Met Arg Lys Tyr Glu Ala Arg Leu Tyr Ile Leu Gln Gln Ala 2350 2355 2360 2365 cga cgg gat cca ctc acc aaa caa att tct gat aac caa ata ctg ctt 7153 Arg Arg Asp Pro Leu Thr Lys Gln Ile Ser Asp Asn Gln Ile Leu Leu 2370 2375 2380 caa gaa ctg ggt cct gga gat ggt atc gtc atg gcg ttc gat aac gtc 7201 Gln Glu Leu Gly Pro Gly Asp Gly Ile Val Met Ala Phe Asp Asn Val 2385 2390 2395 ctg cag aaa ctc ctg gag gaa tat ggg agt gat gac aca agg aat gtg 7249 Leu Gln Lys Leu Leu Glu Glu Tyr Gly Ser Asp Asp Thr Arg Asn Val 2400 2405 2410 aaa gaa acc aca gag tac tta aaa aca tca tgg atc aat ctc aaa caa 7297 Lys Glu Thr Thr Glu Tyr Leu Lys Thr Ser Trp Ile Asn Leu Lys Gln 2415 2420 2425 agt att gct gac aga cag aac gcc ttg gag gct gag tgg agg acg gtg 7345 Ser Ile Ala Asp Arg Gln Asn Ala Leu Glu Ala Glu Trp Arg Thr Val 2430 2435 2440 2445 cag gcc tct cgc aga gat ctg gaa aac ttc ctg aag tgg atc caa gaa 7393 Gln Ala Ser Arg Arg Asp Leu Glu Asn Phe Leu Lys Trp Ile Gln Glu 2450 2455 2460 gca gag acc aca gtg aat gtg ctt gtg gat gcc tct cat cgg gag aat 7441 Ala Glu Thr Thr Val Asn Val Leu Val Asp Ala Ser His Arg Glu Asn 2465 2470 2475 gct ctt cag gat agt atc ttg gcc agg gaa ctc aaa cag cag atg cag 7489 Ala Leu Gln Asp Ser Ile Leu Ala Arg Glu Leu Lys Gln Gln Met Gln 2480 2485 2490 gac atc cag gca gaa att gat gcc cac aat gac ata ttt aaa agc att 7537 Asp Ile Gln Ala Glu Ile Asp Ala His Asn Asp Ile Phe Lys Ser Ile 2495 2500 2505 gac gga aac agg cag aag atg gta aaa gct ttg gga aat tct gaa gag 7585 Asp Gly Asn Arg Gln Lys Met Val Lys Ala Leu Gly Asn Ser Glu Glu 2510 2515 2520 2525 gct act atg ctt caa cat cga ctg gat gat atg aac caa aga tgg aat 7633 Ala Thr Met Leu Gln His Arg Leu Asp Asp Met Asn Gln Arg Trp Asn 2530 2535 2540 gac tta aaa gca aaa tct gct agc atc agg gcc cat ttg gag gcc agc 7681 Asp Leu Lys Ala Lys Ser Ala Ser Ile Arg Ala His Leu Glu Ala Ser 2545 2550 2555 gct gag aag tgg aac agg ttg ctg atg tcc tta gaa gaa ctg atc aaa 7729 Ala Glu Lys Trp Asn Arg Leu Leu Met Ser Leu Glu Glu Leu Ile Lys 2560 2565 2570 tgg ctg aat atg aaa gat gaa gag ctt aag aaa caa atg cct att gga 7777 Trp Leu Asn Met Lys Asp Glu Glu Leu Lys Lys Gln Met Pro Ile Gly 2575 2580 2585 gga gat gtt cca gcc tta cag ctc cag tat gac cat tgt aag gcc ctg 7825 Gly Asp Val Pro Ala Leu Gln Leu Gln Tyr Asp His Cys Lys Ala Leu 2590 2595 2600 2605 aga cgg gag tta aag gag aaa gaa tat tct gtc ctg aat gct gtc gac 7873 Arg Arg Glu Leu Lys Glu Lys Glu Tyr Ser Val Leu Asn Ala Val Asp 2610 2615 2620 cag gcc cga gtt ttc ttg gct gat cag cca att gag gcc cct gaa gag 7921 Gln Ala Arg Val Phe Leu Ala Asp Gln Pro Ile Glu Ala Pro Glu Glu 2625 2630 2635 cca aga aga aac cta caa tca aaa aca gaa tta act cct gag gag aga 7969 Pro Arg Arg Asn Leu Gln Ser Lys Thr Glu Leu Thr Pro Glu Glu Arg 2640 2645 2650 gcc caa aag att gcc aaa gcc atg cgc aaa cag tct tct gaa gtc aaa 8017 Ala Gln Lys Ile Ala Lys Ala Met Arg Lys Gln Ser Ser Glu Val Lys 2655 2660 2665 gaa aaa tgg gaa agt cta aat gct gta act agc aat tgg caa aag caa 8065 Glu Lys Trp Glu Ser Leu Asn Ala Val Thr Ser Asn Trp Gln Lys Gln 2670 2675 2680 2685 gtg gac aag gca ttg gag aaa ctc aga gac ctg cag gga gct atg gat 8113 Val Asp Lys Ala Leu Glu Lys Leu Arg Asp Leu Gln Gly Ala Met Asp 2690 2695 2700 gac ctg gac gct gac atg aag gag gca gag tcc gtg cgg aat ggc tgg 8161 Asp Leu Asp Ala Asp Met Lys Glu Ala Glu Ser Val Arg Asn Gly Trp 2705 2710 2715 aag ccc gtg gga gac tta ctc att gac tcg ctg cag gat cac att gaa 8209 Lys Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His Ile Glu 2720 2725 2730 aaa atc atg gca ttt aga gaa gaa att gca cca atc aac ttt aaa gtt 8257 Lys Ile Met Ala Phe Arg Glu Glu Ile Ala Pro Ile Asn Phe Lys Val 2735 2740 2745 aaa acg gtg aat gat tta tcc agt cag ctg tct cca ctt gac ctg cat 8305 Lys Thr Val Asn Asp Leu Ser Ser Gln Leu Ser Pro Leu Asp Leu His 2750 2755 2760 2765 ccc tct cta aag atg tct cgc cag cta gat gac ctt aat atg cga tgg 8353 Pro Ser Leu Lys Met Ser Arg Gln Leu Asp Asp Leu Asn Met Arg Trp 2770 2775 2780 aaa ctt tta cag gtt tct gtg gat gat cgc ctt aaa cag ctt cag gaa 8401 Lys Leu Leu Gln Val Ser Val Asp Asp Arg Leu Lys Gln Leu Gln Glu 2785 2790 2795 gcc cac aga gat ttt gga cca tcc tct cag cat ttt ctc tct acg tca 8449 Ala His Arg Asp Phe Gly Pro Ser Ser Gln His Phe Leu Ser Thr Ser 2800 2805 2810 gtc cag ctg ccg tgg caa aga tcc att tca cat aat aaa gtg ccc tat 8497 Val Gln Leu Pro Trp Gln Arg Ser Ile Ser His Asn Lys Val Pro Tyr 2815 2820 2825 tac atc aac cat caa aca cag acc acc tgt tgg gac cat cct aaa atg 8545 Tyr Ile Asn His Gln Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met 2830 2835 2840 2845 acc gaa ctc ttt caa tcc ctt gct gac ctg aat aat gta cgt ttt tct 8593 Thr Glu Leu Phe Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser 2850 2855 2860 gcc tac cgt aca gca atc aaa atc cga aga cta caa aaa gca cta tgt 8641 Ala Tyr Arg Thr Ala Ile Lys Ile Arg Arg Leu Gln Lys Ala Leu Cys 2865 2870 2875 ttg gat ctc tta gag ttg agt aca aca aat gaa att ttc aaa cag cac 8689 Leu Asp Leu Leu Glu Leu Ser Thr Thr Asn Glu Ile Phe Lys Gln His 2880 2885 2890 aag ttg aac caa aat gac cag ctc ctc agt gtt cca gat gtc atc aac 8737 Lys Leu Asn Gln Asn Asp Gln Leu Leu Ser Val Pro Asp Val Ile Asn 2895 2900 2905 tgt ctg aca aca act tat gat gga ctt gag caa atg cat aag gac ctg 8785 Cys Leu Thr Thr Thr Tyr Asp Gly Leu Glu Gln Met His Lys Asp Leu 2910 2915 2920 2925 gtc aac gtt cca ctc tgt gtt gat atg tgt ctc aat tgg ttg ctc aat 8833 Val Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn 2930 2935 2940 gtc tat gac acg ggt cga act gga aaa att aga gtg cag agt ctg aag 8881 Val Tyr Asp Thr Gly Arg Thr Gly Lys Ile Arg Val Gln Ser Leu Lys 2945 2950 2955 att gga tta atg tct ctc tcc aaa ggt ctc ttg gaa gaa aaa tac aga 8929 Ile Gly Leu Met Ser Leu Ser Lys Gly Leu Leu Glu Glu Lys Tyr Arg 2960 2965 2970 tat ctc ttt aag gaa gtt gcg ggg ccg aca gaa atg tgt gac cag agg 8977 Tyr Leu Phe Lys Glu Val Ala Gly Pro Thr Glu Met Cys Asp Gln Arg 2975 2980 2985 cag ctg ggc ctg tta ctt cat gat gcc atc cag atc ccc cgg cag cta 9025 Gln Leu Gly Leu Leu Leu His Asp Ala Ile Gln Ile Pro Arg Gln Leu 2990 2995 3000 3005 ggt gaa gta gca gct ttt gga ggc agt aat att gag cct agt gtt cgc 9073 Gly Glu Val Ala Ala Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg 3010 3015 3020 agc tgc ttc caa cag aat aac aat aaa cca gaa ata agt gtg aaa gag 9121 Ser Cys Phe Gln Gln Asn Asn Asn Lys Pro Glu Ile Ser Val Lys Glu 3025 3030 3035 ttt ata gat tgg atg cat ttg gaa cca cag tcc atg gtt tgg ctc cca 9169 Phe Ile Asp Trp Met His Leu Glu Pro Gln Ser Met Val Trp Leu Pro 3040 3045 3050 gtt tta cat cga gtg gca gca gcg gag act gca aaa cat cag gcc aaa 9217 Val Leu His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys 3055 3060 3065 tgc aac atc tgt aaa gaa tgt cca att gtc ggg ttc agg tat aga agc 9265 Cys Asn Ile Cys Lys Glu Cys Pro Ile Val Gly Phe Arg Tyr Arg Ser 3070 3075 3080 3085 ctt aag cat ttt aac tat gat gtc tgc cag agt tgt ttc ttt tcg ggt 9313 Leu Lys His Phe Asn Tyr Asp Val Cys Gln Ser Cys Phe Phe Ser Gly 3090 3095 3100 cga aca gca aaa ggt cac aaa tta cat tac cca atg gtg gaa tat tgt 9361 Arg Thr Ala Lys Gly His Lys Leu His Tyr Pro Met Val Glu Tyr Cys 3105 3110 3115 ata cct aca aca tct ggg gaa gat gta cga gac ttc aca aag gta ctt 9409 Ile Pro Thr Thr Ser Gly Glu Asp Val Arg Asp Phe Thr Lys Val Leu 3120 3125 3130 aag aac aag ttc agg tcg aag aag tac ttt gcc aaa cac cct cga ctt 9457 Lys Asn Lys Phe Arg Ser Lys Lys Tyr Phe Ala Lys His Pro Arg Leu 3135 3140 3145 ggt tac ctg cct gtc cag aca gtt ctt gaa ggt gac aac tta gag act 9505 Gly Tyr Leu Pro Val Gln Thr Val Leu Glu Gly Asp Asn Leu Glu Thr 3150 3155 3160 3165 cct atc aca ctc atc agt atg tgg cca gag cac tat gac ccc tca caa 9553 Pro Ile Thr Leu Ile Ser Met Trp Pro Glu His Tyr Asp Pro Ser Gln 3170 3175 3180 tct cct caa ctg ttt cat gat gac acc cat tca aga ata gaa caa tat 9601 Ser Pro Gln Leu Phe His Asp Asp Thr His Ser Arg Ile Glu Gln Tyr 3185 3190 3195 gcc aca cga ctg gcc cag atg gaa agg act aat ggg tct ttt ctc act 9649 Ala Thr Arg Leu Ala Gln Met Glu Arg Thr Asn Gly Ser Phe Leu Thr 3200 3205 3210 gat agc agc tcc acc aca gga agt gtg gaa gac gag cac gcc ctc atc 9697 Asp Ser Ser Ser Thr Thr Gly Ser Val Glu Asp Glu His Ala Leu Ile 3215 3220 3225 cag cag tat tgc caa aca ctc gga gga gag tcc cca gtg agc cag ccg 9745 Gln Gln Tyr Cys Gln Thr Leu Gly Gly Glu Ser Pro Val Ser Gln Pro 3230 3235 3240 3245 cag agc cca gct cag atc ctg aag tca gta gag agg gaa gaa cgt gga 9793 Gln Ser Pro Ala Gln Ile Leu Lys Ser Val Glu Arg Glu Glu Arg Gly 3250 3255 3260 gaa ctg gag agg atc att gct gac ctg gag gaa gaa caa aga aat cta 9841 Glu Leu Glu Arg Ile Ile Ala Asp Leu Glu Glu Glu Gln Arg Asn Leu 3265 3270 3275 cag gtg gag tat gag cag ctg aag gac cag cac ctc cga agg ggg ctc 9889 Gln Val Glu Tyr Glu Gln Leu Lys Asp Gln His Leu Arg Arg Gly Leu 3280 3285 3290 cct gtc ggt tca ccg cca gag tcg att ata tct ccc cat cac acg tct 9937 Pro Val Gly Ser Pro Pro Glu Ser Ile Ile Ser Pro His His Thr Ser 3295 3300 3305 gag gat tca gaa ctt ata gca gaa gca aaa ctc ctc agg cag cac aaa 9985 Glu Asp Ser Glu Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys 3310 3315 3320 3325 ggt cgg ctg gag gct agg atg cag att tta gaa gat cac aat aaa cag 10033 Gly Arg Leu Glu Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys Gln 3330 3335 3340 ctg gag tct cag ctc cac cgc ctc cga cag ctg ctg gag cag cct gaa 10081 Leu Glu Ser Gln Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Glu 3345 3350 3355 tct gat tcc cga atc aat ggt gtt tcc cca tgg gct tct cct cag cat 10129 Ser Asp Ser Arg Ile Asn Gly Val Ser Pro Trp Ala Ser Pro Gln His 3360 3365 3370 tct gca ctg agc tac tcg ctt gat cca gat gcc tcc ggc cca cag ttc 10177 Ser Ala Leu Ser Tyr Ser Leu Asp Pro Asp Ala Ser Gly Pro Gln Phe 3375 3380 3385 cac cag gca gcg gga gag gac ctg ctg gcc cca ccg cac gac acc agc 10225 His Gln Ala Ala Gly Glu Asp Leu Leu Ala Pro Pro His Asp Thr Ser 3390 3395 3400 3405 acg gat ctc acg gag gtc atg gag cag att cac agc acg ttt cca tct 10273 Thr Asp Leu Thr Glu Val Met Glu Gln Ile His Ser Thr Phe Pro Ser 3410 3415 3420 tgc tgc cca aat gtt ccc agc agg cca cag gca atg taa tcactagt 10320 Cys Cys Pro Asn Val Pro Ser Arg Pro Gln Ala Met 3425 3430 10 3433 PRT Artificial Sequence misc_feature (239) ... (250) Description of Artificial Sequence Full length utrophin construct; Xaa = unknown 10 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn Gly Gln Asn 1 5 10 15 Glu Phe Ser Asp Ile Ile Glu Ser Arg Ser Asp Glu His Asn Asp Val 20 25 30 Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe Ser Lys Ser 35 40 45 Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys Asp Gly Arg 50 55 60 Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser Leu Pro Lys 65 70 75 80 Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Arg Val 85 90 95 Leu Gln Val Leu His Gln Asn Asn Val Asp Leu Val Asn Ile Gly Gly 100 105 110 Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly Leu Leu Trp 115 120 125 Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys Asp Ile Met 130 135 140 Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 145 150 155 160 Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu Asn Phe Thr 165 170 175 Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu His Arg His 180 185 190 Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met Ser Pro Ile 195 200 205 Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr Leu Gly Ile 210 215 220 Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val His Leu Pro Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Glu Val Leu Pro Gln 245 250 255 Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu Pro Arg Lys 260 265 270 Tyr Lys Lys Glu Cys Glu Glu Glu Glu Ile His Ile Gln Ser Ala Val 275 280 285 Leu Ala Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr Pro Ser Thr Val 290 295 300 Thr Glu Val Asp Met Asp Leu Asp Ser Tyr Gln Ile Ala Leu Glu Glu 305 310 315 320 Val Leu Thr Trp Leu Leu Ser Ala Glu Asp Thr Phe Gln Glu Gln Asp 325 330 335 Asp Ile Ser Asp Asp Val Glu Glu Val Lys Glu Gln Phe Ala Thr His 340 345 350 Glu Thr Phe Met Met Glu Leu Thr Ala His Gln Ser Ser Val Gly Ser 355 360 365 Val Leu Gln Ala Gly Asn Gln Leu Met Thr Gln Gly Thr Leu Ser Glu 370 375 380 Glu Glu Glu Phe Glu Ile Gln Glu Gln Met Thr Leu Leu Asn Ala Arg 385 390 395 400 Trp Glu Ala Leu Arg Val Glu Ser Met Glu Arg Gln Ser Arg Leu His 405 410 415 Asp Ala Leu Met Glu Leu Gln Lys Lys Gln Leu Gln Gln Leu Ser Ser 420 425 430 Trp Leu Ala Leu Thr Glu Glu Arg Ile Gln Lys Met Glu Ser Pro Pro 435 440 445 Leu Gly Asp Asp Leu Pro Ser Leu Gln Lys Leu Leu Gln Glu His Lys 450 455 460 Ser Leu Gln Asn Asp Leu Glu Ala Glu Gln Val Lys Val Asn Ser Leu 465 470 475 480 Thr His Met Val Val Ile Val Asp Glu Asn Ser Gly Glu Ser Ala Thr 485 490 495 Ala Leu Leu Glu Asp Gln Leu Gln Lys Leu Gly Glu Arg Trp Thr Ala 500 505 510 Val Cys Arg Trp Thr Glu Glu Arg Trp Asn Arg Leu Gln Glu Ile Ser 515 520 525 Ile Leu Trp Gln Glu Leu Leu Glu Glu Gln Cys Leu Leu Glu Ala Trp 530 535 540 Leu Thr Glu Lys Glu Glu Ala Leu Asn Lys Val Gln Thr Ser Asn Phe 545 550 555 560 Lys Asp Gln Lys Glu Leu Ser Val Ser Val Arg Arg Leu Ala Ile Leu 565 570 575 Lys Glu Asp Met Glu Met Lys Arg Gln Thr Leu Asp Gln Leu Ser Glu 580 585 590 Ile Gly Gln Asp Val Gly Gln Leu Leu Ser Asn Pro Lys Ala Ser Lys 595 600 605 Lys Met Asn Ser Asp Ser Glu Glu Leu Thr Gln Arg Trp Asp Ser Leu 610 615 620 Val Gln Arg Leu Glu Asp Ser Ser Asn Gln Val Thr Gln Ala Val Ala 625 630 635 640 Lys Leu Gly Met Ser Gln Ile Pro Gln Lys Asp Leu Leu Glu Thr Val 645 650 655 His Val Arg Glu Lys Gly Met Val Lys Lys Pro Lys Gln Glu Leu Pro 660 665 670 Pro Pro Leu Gly Pro Lys Lys Arg Gln Ile His Val Asp Ile Glu Ala 675 680 685 Lys Lys Lys Phe Asp Ala Ile Ser Ala Glu Leu Leu Asn Trp Ile Leu 690 695 700 Lys Trp Lys Thr Ala Ile Gln Thr Thr Glu Ile Lys Glu Tyr Met Lys 705 710 715 720 Met Gln Asp Thr Ser Glu Met Lys Lys Lys Leu Lys Ala Leu Glu Lys 725 730 735 Glu Gln Arg Glu Arg Ile Pro Arg Ala Asp Glu Leu Asn Gln Thr Gly 740 745 750 Gln Ile Leu Val Glu Gln Met Gly Lys Glu Gly Leu Pro Thr Glu Glu 755 760 765 Ile Lys Asn Val Leu Glu Lys Val Ser Ser Glu Trp Lys Asn Val Ser 770 775 780 Gln His Leu Glu Asp Leu Glu Arg Lys Ile Gln Leu Gln Glu Asp Ile 785 790 795 800 Asn Ala Tyr Phe Lys Gln Leu Asp Glu Leu Glu Lys Val Ile Lys Thr 805 810 815 Lys Glu Glu Trp Val Lys His Thr Ser Ile Ser Glu Ser Ser Arg Gln 820 825 830 Ser Leu Pro Ser Leu Lys Asp Ser Cys Gln Arg Glu Leu Thr Asn Leu 835 840 845 Leu Gly Leu His Pro Lys Ile Glu Met Ala Arg Ala Ser Cys Ser Ala 850 855 860 Leu Met Ser Gln Pro Ser Ala Pro Asp Phe Val Gln Arg Gly Phe Asp 865 870 875 880 Ser Phe Leu Gly Arg Tyr Gln Ala Val Gln Glu Ala Val Glu Asp Arg 885 890 895 Gln Gln His Leu Glu Asn Glu Leu Lys Gly Gln Pro Gly His Ala Tyr 900 905 910 Leu Glu Thr Leu Lys Thr Leu Lys Asp Val Leu Asn Asp Ser Glu Asn 915 920 925 Lys Ala Gln Val Ser Leu Asn Val Leu Asn Asp Leu Ala Lys Val Glu 930 935 940 Lys Ala Leu Gln Glu Lys Lys Thr Leu Asp Glu Ile Leu Glu Asn Gln 945 950 955 960 Lys Pro Ala Leu His Lys Leu Ala Glu Glu Thr Lys Ala Leu Glu Lys 965 970 975 Asn Val His Pro Asp Val Glu Lys Leu Tyr Lys Gln Glu Phe Asp Asp 980 985 990 Val Gln Gly Lys Trp Asn Lys Leu Lys Val Leu Val Ser Lys Asp Leu 995 1000 1005 His Leu Leu Glu Glu Ile Ala Leu Thr Leu Arg Ala Phe Glu Ala Asp 1010 1015 1020 Ser Thr Val Ile Glu Lys Trp Met Asp Gly Val Lys Asp Phe Leu Met 1025 1030 1035 1040 Lys Gln Gln Ala Ala Gln Gly Asp Asp Ala Gly Leu Gln Arg Gln Leu 1045 1050 1055 Asp Gln Cys Ser Ala Phe Val Asn Glu Ile Glu Thr Ile Glu Ser Ser 1060 1065 1070 Leu Lys Asn Met Lys Glu Ile Glu Thr Asn Leu Arg Ser Gly Pro Val 1075 1080 1085 Ala Gly Ile Lys Thr Trp Val Gln Thr Arg Leu Gly Asp Tyr Gln Thr 1090 1095 1100 Gln Leu Glu Lys Leu Ser Lys Glu Ile Ala Thr Gln Lys Ser Arg Leu 1105 1110 1115 1120 Ser Glu Ser Gln Glu Lys Ala Ala Asn Leu Lys Lys Asp Leu Ala Glu 1125 1130 1135 Met Gln Glu Trp Met Thr Gln Ala Glu Glu Glu Tyr Leu Glu Arg Asp 1140 1145 1150 Phe Glu Tyr Lys Ser Pro Glu Glu Leu Glu Ser Ala Val Glu Glu Met 1155 1160 1165 Lys Arg Ala Lys Glu Asp Val Leu Gln Lys Glu Val Arg Val Lys Ile 1170 1175 1180 Leu Lys Asp Asn Ile Lys Leu Leu Ala Ala Lys Val Pro Ser Gly Gly 1185 1190 1195 1200 Gln Glu Leu Thr Ser Glu Leu Asn Val Val Leu Glu Asn Tyr Gln Leu 1205 1210 1215 Leu Cys Asn Arg Ile Arg Gly Lys Cys His Thr Leu Glu Glu Val Trp 1220 1225 1230 Ser Cys Trp Ile Glu Leu Leu His Tyr Leu Asp Leu Glu Thr Thr Trp 1235 1240 1245 Leu Asn Thr Leu Glu Glu Arg Met Lys Ser Thr Glu Val Leu Pro Glu 1250 1255 1260 Lys Thr Asp Ala Val Asn Glu Ala Leu Glu Ser Leu Glu Ser Val Leu 1265 1270 1275 1280 Arg His Pro Ala Asp Asn Arg Thr Gln Ile Arg Glu Leu Gly Gln Thr 1285 1290 1295 Leu Ile Asp Gly Gly Ile Leu Asp Asp Ile Ile Ser Glu Lys Leu Glu 1300 1305 1310 Ala Phe Asn Ser Arg Tyr Glu Asp Leu Ser His Leu Ala Glu Ser Lys 1315 1320 1325 Gln Ile Ser Leu Glu Lys Gln Leu Gln Val Leu Arg Glu Thr Asp Gln 1330 1335 1340 Met Leu Gln Val Leu Gln Glu Ser Leu Gly Glu Leu Asp Lys Gln Leu 1345 1350 1355 1360 Thr Thr Tyr Leu Thr Asp Arg Ile Asp Ala Phe Gln Val Pro Gln Glu 1365 1370 1375 Ala Gln Lys Ile Gln Ala Glu Ile Ser Ala His Glu Leu Thr Leu Glu 1380 1385 1390 Glu Leu Arg Arg Asn Met Arg Ser Gln Pro Leu Thr Ser Pro Glu Ser 1395 1400 1405 Arg Thr Ala Arg Gly Gly Ser Gln Met Asp Val Leu Gln Arg Lys Leu 1410 1415 1420 Arg Glu Val Ser Thr Lys Phe Gln Leu Phe Gln Lys Pro Ala Asn Phe 1425 1430 1435 1440 Glu Gln Arg Met Leu Asp Cys Lys Arg Val Leu Asp Gly Val Lys Ala 1445 1450 1455 Glu Leu His Val Leu Asp Val Lys Asp Val Asp Pro Asp Val Ile Gln 1460 1465 1470 Thr His Leu Asp Lys Cys Met Lys Leu Tyr Lys Thr Leu Ser Glu Val 1475 1480 1485 Lys Leu Glu Val Glu Thr Val Ile Lys Thr Gly Arg His Ile Val Gln 1490 1495 1500 Lys Gln Gln Thr Asp Asn Pro Lys Gly Met Asp Glu Gln Leu Thr Ser 1505 1510 1515 1520 Leu Lys Val Leu Tyr Asn Asp Leu Gly Ala Gln Val Thr Glu Gly Lys 1525 1530 1535 Gln Asp Leu Glu Arg Ala Ser Gln Leu Ala Arg Lys Met Lys Lys Glu 1540 1545 1550 Ala Ala Ser Leu Ser Glu Trp Leu Ser Ala Thr Glu Thr Glu Leu Val 1555 1560 1565 Gln Lys Ser Thr Ser Glu Gly Leu Leu Gly Asp Leu Asp Thr Glu Ile 1570 1575 1580 Ser Trp Ala Lys Asn Val Leu Lys Asp Leu Glu Lys Arg Lys Ala Asp 1585 1590 1595 1600 Leu Asn Thr Ile Thr Glu Ser Ser Ala Ala Leu Gln Asn Leu Ile Glu 1605 1610 1615 Gly Ser Glu Pro Ile Leu Glu Glu Arg Leu Cys Val Leu Asn Ala Gly 1620 1625 1630 Trp Ser Arg Val Arg Thr Trp Thr Glu Asp Trp Cys Asn Thr Leu Met 1635 1640 1645 Asn His Gln Asn Gln Leu Glu Ile Phe Asp Gly Asn Val Ala His Ile 1650 1655 1660 Ser Thr Trp Leu Tyr Gln Ala Glu Ala Leu Leu Asp Glu Ile Glu Lys 1665 1670 1675 1680 Lys Pro Thr Ser Lys Gln Glu Glu Ile Val Lys Arg Leu Val Ser Glu 1685 1690 1695 Leu Asp Asp Ala Asn Leu Gln Val Glu Asn Val Arg Asp Gln Ala Leu 1700 1705 1710 Ile Leu Met Asn Ala Arg Gly Ser Ser Ser Arg Glu Leu Val Glu Pro 1715 1720 1725 Lys Leu Ala Glu Leu Asn Arg Asn Phe Glu Lys Val Ser Gln His Ile 1730 1735 1740 Lys Ser Ala Lys Leu Leu Ile Ala Gln Glu Pro Leu Tyr Gln Cys Leu 1745 1750 1755 1760 Val Thr Thr Glu Thr Phe Glu Thr Gly Val Pro Phe Ser Asp Leu Glu 1765 1770 1775 Lys Leu Glu Asn Asp Ile Glu Asn Met Leu Lys Phe Val Glu Lys His 1780 1785 1790 Leu Glu Ser Ser Asp Glu Asp Glu Lys Met Asp Glu Glu Ser Ala Gln 1795 1800 1805 Ile Glu Glu Val Leu Gln Arg Gly Glu Glu Met Leu His Gln Pro Met 1810 1815 1820 Glu Asp Asn Lys Lys Glu Lys Ile Arg Leu Gln Leu Leu Leu Leu His 1825 1830 1835 1840 Thr Arg Tyr Asn Lys Ile Lys Ala Ile Pro Ile Gln Gln Arg Lys Met 1845 1850 1855 Gly Gln Leu Ala Ser Gly Ile Arg Ser Ser Leu Leu Pro Thr Asp Tyr 1860 1865 1870 Leu Val Glu Ile Asn Lys Ile Leu Leu Cys Met Asp Asp Val Glu Leu 1875 1880 1885 Ser Leu Asn Val Pro Glu Leu Asn Thr Ala Ile Tyr Glu Asp Phe Ser 1890 1895 1900 Phe Gln Glu Asp Ser Leu Lys Asn Ile Lys Asp Gln Leu Asp Lys Leu 1905 1910 1915 1920 Gly Glu Gln Ile Ala Val Ile His Glu Lys Gln Pro Asp Val Ile Leu 1925 1930 1935 Glu Ala Ser Gly Pro Glu Ala Ile Gln Ile Arg Asp Thr Leu Thr Gln 1940 1945 1950 Leu Asn Ala Lys Trp Asp Arg Ile Asn Arg Met Tyr Ser Asp Arg Lys 1955 1960 1965 Gly Cys Phe Asp Arg Ala Met Glu Glu Trp Arg Gln Phe His Cys Asp 1970 1975 1980 Leu Asn Asp Leu Thr Gln Trp Ile Thr Glu Ala Glu Glu Leu Leu Val 1985 1990 1995 2000 Asp Thr Cys Ala Pro Gly Gly Ser Leu Asp Leu Glu Lys Ala Arg Ile 2005 2010 2015 His Gln Gln Glu Leu Glu Val Gly Ile Ser Ser His Gln Pro Ser Phe 2020 2025 2030 Ala Ala Leu Asn Arg Thr Gly Asp Gly Ile Val Gln Lys Leu Ser Gln 2035 2040 2045 Ala Asp Gly Ser Phe Leu Lys Glu Lys Leu Ala Gly Leu Asn Gln Arg 2050 2055 2060 Trp Asp Ala Ile Val Ala Glu Val Lys Asp Arg Gln Pro Arg Leu Lys 2065 2070 2075 2080 Gly Glu Ser Lys Gln Val Met Lys Tyr Arg His Gln Leu Asp Glu Ile 2085 2090 2095 Ile Cys Trp Leu Thr Lys Ala Glu His Ala Met Gln Lys Arg Ser Thr 2100 2105 2110 Thr Glu Leu Gly Glu Asn Leu Gln Glu Leu Arg Asp Leu Thr Gln Glu 2115 2120 2125 Met Glu Val His Ala Glu Lys Leu Lys Trp Leu Asn Arg Thr Glu Leu 2130 2135 2140 Glu Met Leu Ser Asp Lys Ser Leu Ser Leu Pro Glu Arg Asp Lys Ile 2145 2150 2155 2160 Ser Glu Ser Leu Arg Thr Val Asn Met Thr Trp Asn Lys Ile Cys Arg 2165 2170 2175 Glu Val Pro Thr Thr Leu Lys Glu Cys Ile Gln Glu Pro Ser Ser Val 2180 2185 2190 Ser Gln Thr Arg Ile Ala Ala His Pro Asn Val Gln Lys Val Val Leu 2195 2200 2205 Val Ser Ser Ala Ser Asp Ile Pro Val Gln Ser His Arg Thr Ser Glu 2210 2215 2220 Ile Ser Ile Pro Ala Asp Leu Asp Lys Thr Ile Thr Glu Leu Ala Asp 2225 2230 2235 2240 Trp Leu Val Leu Ile Asp Gln Met Leu Lys Ser Asn Ile Val Thr Val 2245 2250 2255 Gly Asp Val Glu Glu Ile Asn Lys Thr Val Ser Arg Met Lys Ile Thr 2260 2265 2270 Lys Ala Asp Leu Glu Gln Arg His Pro Gln Leu Asp Tyr Val Phe Thr 2275 2280 2285 Leu Ala Gln Asn Leu Lys Asn Lys Ala Ser Ser Ser Asp Met Arg Thr 2290 2295 2300 Ala Ile Thr Glu Lys Leu Glu Arg Val Lys Asn Gln Trp Asp Gly Thr 2305 2310 2315 2320 Gln His Gly Val Glu Leu Arg Gln Gln Gln Leu Glu Asp Met Ile Ile 2325 2330 2335 Asp Ser Leu Gln Trp Asp Asp His Arg Glu Glu Thr Glu Glu Leu Met 2340 2345 2350 Arg Lys Tyr Glu Ala Arg Leu Tyr Ile Leu Gln Gln Ala Arg Arg Asp 2355 2360 2365 Pro Leu Thr Lys Gln Ile Ser Asp Asn Gln Ile Leu Leu Gln Glu Leu 2370 2375 2380 Gly Pro Gly Asp Gly Ile Val Met Ala Phe Asp Asn Val Leu Gln Lys 2385 2390 2395 2400 Leu Leu Glu Glu Tyr Gly Ser Asp Asp Thr Arg Asn Val Lys Glu Thr 2405 2410 2415 Thr Glu Tyr Leu Lys Thr Ser Trp Ile Asn Leu Lys Gln Ser Ile Ala 2420 2425 2430 Asp Arg Gln Asn Ala Leu Glu Ala Glu Trp Arg Thr Val Gln Ala Ser 2435 2440 2445 Arg Arg Asp Leu Glu Asn Phe Leu Lys Trp Ile Gln Glu Ala Glu Thr 2450 2455 2460 Thr Val Asn Val Leu Val Asp Ala Ser His Arg Glu Asn Ala Leu Gln 2465 2470 2475 2480 Asp Ser Ile Leu Ala Arg Glu Leu Lys Gln Gln Met Gln Asp Ile Gln 2485 2490 2495 Ala Glu Ile Asp Ala His Asn Asp Ile Phe Lys Ser Ile Asp Gly Asn 2500 2505 2510 Arg Gln Lys Met Val Lys Ala Leu Gly Asn Ser Glu Glu Ala Thr Met 2515 2520 2525 Leu Gln His Arg Leu Asp Asp Met Asn Gln Arg Trp Asn Asp Leu Lys 2530 2535 2540 Ala Lys Ser Ala Ser Ile Arg Ala His Leu Glu Ala Ser Ala Glu Lys 2545 2550 2555 2560 Trp Asn Arg Leu Leu Met Ser Leu Glu Glu Leu Ile Lys Trp Leu Asn 2565 2570 2575 Met Lys Asp Glu Glu Leu Lys Lys Gln Met Pro Ile Gly Gly Asp Val 2580 2585 2590 Pro Ala Leu Gln Leu Gln Tyr Asp His Cys Lys Ala Leu Arg Arg Glu 2595 2600 2605 Leu Lys Glu Lys Glu Tyr Ser Val Leu Asn Ala Val Asp Gln Ala Arg 2610 2615 2620 Val Phe Leu Ala Asp Gln Pro Ile Glu Ala Pro Glu Glu Pro Arg Arg 2625 2630 2635 2640 Asn Leu Gln Ser Lys Thr Glu Leu Thr Pro Glu Glu Arg Ala Gln Lys 2645 2650 2655 Ile Ala Lys Ala Met Arg Lys Gln Ser Ser Glu Val Lys Glu Lys Trp 2660 2665 2670 Glu Ser Leu Asn Ala Val Thr Ser Asn Trp Gln Lys Gln Val Asp Lys 2675 2680 2685 Ala Leu Glu Lys Leu Arg Asp Leu Gln Gly Ala Met Asp Asp Leu Asp 2690 2695 2700 Ala Asp Met Lys Glu Ala Glu Ser Val Arg Asn Gly Trp Lys Pro Val 2705 2710 2715 2720 Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His Ile Glu Lys Ile Met 2725 2730 2735 Ala Phe Arg Glu Glu Ile Ala Pro Ile Asn Phe Lys Val Lys Thr Val 2740 2745 2750 Asn Asp Leu Ser Ser Gln Leu Ser Pro Leu Asp Leu His Pro Ser Leu 2755 2760 2765 Lys Met Ser Arg Gln Leu Asp Asp Leu Asn Met Arg Trp Lys Leu Leu 2770 2775 2780 Gln Val Ser Val Asp Asp Arg Leu Lys Gln Leu Gln Glu Ala His Arg 2785 2790 2795 2800 Asp Phe Gly Pro Ser Ser Gln His Phe Leu Ser Thr Ser Val Gln Leu 2805 2810 2815 Pro Trp Gln Arg Ser Ile Ser His Asn Lys Val Pro Tyr Tyr Ile Asn 2820 2825 2830 His Gln Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu 2835 2840 2845 Phe Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser Ala Tyr Arg 2850 2855 2860 Thr Ala Ile Lys Ile Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu 2865 2870 2875 2880 Leu Glu Leu Ser Thr Thr Asn Glu Ile Phe Lys Gln His Lys Leu Asn 2885 2890 2895 Gln Asn Asp Gln Leu Leu Ser Val Pro Asp Val Ile Asn Cys Leu Thr 2900 2905 2910 Thr Thr Tyr Asp Gly Leu Glu Gln Met His Lys Asp Leu Val Asn Val 2915 2920 2925 Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val Tyr Asp 2930 2935 2940 Thr Gly Arg Thr Gly Lys Ile Arg Val Gln Ser Leu Lys Ile Gly Leu 2945 2950 2955 2960 Met Ser Leu Ser Lys Gly Leu Leu Glu Glu Lys Tyr Arg Tyr Leu Phe 2965 2970 2975 Lys Glu Val Ala Gly Pro Thr Glu Met Cys Asp Gln Arg Gln Leu Gly 2980 2985 2990 Leu Leu Leu His Asp Ala Ile Gln Ile Pro Arg Gln Leu Gly Glu Val 2995 3000 3005 Ala Ala Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser Cys Phe 3010 3015 3020 Gln Gln Asn Asn Asn Lys Pro Glu Ile Ser Val Lys Glu Phe Ile Asp 3025 3030 3035 3040 Trp Met His Leu Glu Pro Gln Ser Met Val Trp Leu Pro Val Leu His 3045 3050 3055 Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys Asn Ile 3060 3065 3070 Cys Lys Glu Cys Pro Ile Val Gly Phe Arg Tyr Arg Ser Leu Lys His 3075 3080 3085 Phe Asn Tyr Asp Val Cys Gln Ser Cys Phe Phe Ser Gly Arg Thr Ala 3090 3095 3100 Lys Gly His Lys Leu His Tyr Pro Met Val Glu Tyr Cys Ile Pro Thr 3105 3110 3115 3120 Thr Ser Gly Glu Asp Val Arg Asp Phe Thr Lys Val Leu Lys Asn Lys 3125 3130 3135 Phe Arg Ser Lys Lys Tyr Phe Ala Lys His Pro Arg Leu Gly Tyr Leu 3140 3145 3150 Pro Val Gln Thr Val Leu Glu Gly Asp Asn Leu Glu Thr Pro Ile Thr 3155 3160 3165 Leu Ile Ser Met Trp Pro Glu His Tyr Asp Pro Ser Gln Ser Pro Gln 3170 3175 3180 Leu Phe His Asp Asp Thr His Ser Arg Ile Glu Gln Tyr Ala Thr Arg 3185 3190 3195 3200 Leu Ala Gln Met Glu Arg Thr Asn Gly Ser Phe Leu Thr Asp Ser Ser 3205 3210 3215 Ser Thr Thr Gly Ser Val Glu Asp Glu His Ala Leu Ile Gln Gln Tyr 3220 3225 3230 Cys Gln Thr Leu Gly Gly Glu Ser Pro Val Ser Gln Pro Gln Ser Pro 3235 3240 3245 Ala Gln Ile Leu Lys Ser Val Glu Arg Glu Glu Arg Gly Glu Leu Glu 3250 3255 3260 Arg Ile Ile Ala Asp Leu Glu Glu Glu Gln Arg Asn Leu Gln Val Glu 3265 3270 3275 3280 Tyr Glu Gln Leu Lys Asp Gln His Leu Arg Arg Gly Leu Pro Val Gly 3285 3290 3295 Ser Pro Pro Glu Ser Ile Ile Ser Pro His His Thr Ser Glu Asp Ser 3300 3305 3310 Glu Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu 3315 3320 3325 Glu Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu Glu Ser 3330 3335 3340 Gln Leu His Arg Leu Arg Gln Leu Leu Glu Gln Pro Glu Ser Asp Ser 3345 3350 3355 3360 Arg Ile Asn Gly Val Ser Pro Trp Ala Ser Pro Gln His Ser Ala Leu 3365 3370 3375 Ser Tyr Ser Leu Asp Pro Asp Ala Ser Gly Pro Gln Phe His Gln Ala 3380 3385 3390 Ala Gly Glu Asp Leu Leu Ala Pro Pro His Asp Thr Ser Thr Asp Leu 3395 3400 3405 Thr Glu Val Met Glu Gln Ile His Ser Thr Phe Pro Ser Cys Cys Pro 3410 3415 3420 Asn Val Pro Ser Arg Pro Gln Ala Met 3425 3430 11 300 PRT Homo sapiens 11 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn Gly Gln Asn 1 5 10 15 Glu Phe Ser Asp Ile Ile Lys Ser Arg Ser Asp Glu His Asn Asp Val 20 25 30 Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe Ser Lys Ser 35 40 45 Gly Lys Pro Pro Ile Asn Asp Met Phe Thr Asp Leu Lys Asp Gly Arg 50 55 60 Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser Leu Pro Lys 65 70 75 80 Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Arg Val 85 90 95 Leu Gln Val Leu His Gln Asn Asn Val Glu Leu Val Asn Ile Gly Gly 100 105 110 Thr Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Leu Trp 115 120 125 Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys Asp Ile Met 130 135 140 Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 145 150 155 160 Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu Asn Phe Thr 165 170 175 Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu His Arg His 180 185 190 Lys Pro Asp Leu Phe Ser Trp Asp Lys Val Val Lys Met Ser Pro Ile 195 200 205 Glu Arg Leu Glu His Ala Phe Ser Lys Ala Gln Thr Tyr Leu Gly Ile 210 215 220 Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val Arg Leu Pro Asp Lys 225 230 235 240 Lys Ser Ile Ile Met Tyr Leu Thr Ser Leu Phe Glu Val Leu Pro Gln 245 250 255 Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu Pro Arg Lys 260 265 270 Tyr Lys Lys Glu Cys Glu Glu Glu Ala Ile Asn Ile Gln Ser Thr Ala 275 280 285 Pro Glu Glu Glu His Glu Ser Pro Arg Ala Glu Thr 290 295 300 12 300 PRT Mus sp. SITE (239)..(250) Precise residue is left open 12 Met Ala Lys Tyr Gly Glu His Glu Ala Ser Pro Asp Asn Gly Gln Asn 1 5 10 15 Glu Phe Ser Asp Ile Ile Glu Ser Arg Ser Asp Glu His Asn Asp Val 20 25 30 Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe Ser Lys Ser 35 40 45 Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys Asp Gly Arg 50 55 60 Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser Leu Pro Lys 65 70 75 80 Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Arg Val 85 90 95 Leu Gln Val Leu His Gln Asn Asn Val Asp Leu Val Asn Ile Gly Gly 100 105 110 Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly Leu Leu Trp 115 120 125 Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys Asp Ile Met 130 135 140 Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 145 150 155 160 Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu Asn Phe Thr 165 170 175 Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu His Arg His 180 185 190 Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met Ser Pro Ile 195 200 205 Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr Leu Gly Ile 210 215 220 Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val His Leu Pro Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Glu Val Leu Pro Gln 245 250 255 Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu Pro Arg Lys 260 265 270 Tyr Lys Lys Glu Cys Glu Glu Glu Glu Ile His Ile Gln Ser Ala Val 275 280 285 Leu Ala Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr 290 295 300 13 300 PRT Rattus sp. 13 Met Ala Lys Tyr Gly His Leu Glu Ala Ser Pro Asp Asp Gly Gln Asn 1 5 10 15 Gln Phe Ser Asp Ile Ile Lys Ser Arg Ser Asp Glu His Asn Asp Val 20 25 30 Gln Lys Lys Thr Phe Thr Lys Trp Ile Asn Ala Arg Phe Ser Lys Ser 35 40 45 Gly Lys Pro Pro Ile Asn Asp Met Phe Ser Asp Leu Lys Asp Gly Arg 50 55 60 Lys Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Thr Ser Leu Pro Lys 65 70 75 80 Glu Arg Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Arg Val 85 90 95 Leu Gln Val Leu His Gln Asn Asn Val Glu Leu Val Asn Ile Gly Gly 100 105 110 Thr Asp Ile Val Asp Gly Asn Pro Lys Leu Thr Leu Gly Leu Leu Trp 115 120 125 Ser Ile Ile Leu His Trp Gln Val Lys Asp Val Met Lys Asp Ile Met 130 135 140 Ser Asp Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val 145 150 155 160 Arg Gln Thr Thr Arg Pro Tyr Ser Gln Val Asn Val Leu Asn Phe Thr 165 170 175 Thr Ser Trp Thr Asp Gly Leu Ala Phe Asn Ala Val Leu His Arg His 180 185 190 Lys Pro Asp Leu Phe Ser Trp Asp Arg Val Val Lys Met Ser Pro Thr 195 200 205 Glu Arg Leu Glu His Ala Phe Ser Lys Ala His Thr Tyr Leu Gly Ile 210 215 220 Glu Lys Leu Leu Asp Pro Glu Asp Val Ala Val Gln Leu Pro Asp Lys 225 230 235 240 Lys Ser Ile Ile Met Tyr Leu Thr Ser Leu Phe Glu Val Leu Pro Gln 245 250 255 Gln Val Thr Ile Asp Ala Ile Arg Glu Val Glu Thr Leu Pro Arg Lys 260 265 270 Tyr Lys Lys Glu Cys Glu Gly Glu Glu Ile Asn Ile Gln Ser Ala Val 275 280 285 Leu Thr Glu Glu Gly Gln Ser Pro Arg Ala Glu Thr 290 295 300 14 22 DNA Mus sp. 14 gattgtggat gaaaacagtg gg 22 15 12 PRT Homo sapiens 15 Asp Lys Lys Ser Ile Ile Met Tyr Leu Thr Ser Leu 1 5 10 

What is claimed is:
 1. An isolated nucleic acid encoding the polypeptide with the amino acid sequence of FIG. 3 (SEQ ID NO:8) and which on expression in a host cell produces the polypeptide.
 2. The isolated nucleic acid according to claim 1 wherein the nucleotide sequence of the nucleic acid encoding the polypeptide with the amino acid sequence of FIG. 3 (SEQ ID NO:8) is the coding nucleotide sequence shown in FIG. 3 (SEQ ID NO:7).
 3. The isolated nucleic acid according to claim 1 wherein the nucleotide sequence of the nucleic acid encoding the polypeptide with the amino acid sequence of FIG. 3 (SEQ ID NO:8) differs from the coding nucleotide sequence shown in FIG. 3 (SEQ ID NO:7).
 4. A vector comprising the nucleic acid according to claim
 1. 5. The vector according to claim 4 wherein said vector is an expression vector.
 6. A composition including the nucleic acid according to claim 1 and a pharmaceutically acceptable excipient.
 7. A cell containing the nucleic acid according to claim 1, which nucleic acid is heterologous to the cell.
 8. A cell according to claim 7 which is a muscle cell.
 9. A cell according to claim 7 wherein said polypeptide is expressed.
 10. A method comprising introducing the nucleic acid according to claim 1 into a cell.
 11. A method according to claim 10 wherein said cell is a muscle cell.
 12. A method according to claim 10 comprising introducing said nucleic acid into said cell in vitro.
 13. A method which comprises expressing the coding nucleotide sequence of the nucleic acid according to claim 1 in a cell, which nucleic acid is heterologous to the cell.
 14. The method according to claim 13 wherein said polypeptide is produced, and is then purified or isolated.
 15. The method according to claim 14 wherein the polypeptide is formulated into a composition which comprises a pharmaceutically acceptable excipient, following purification or isolation of the polypeptide.
 16. A method for treating muscular dystrophy in a mammal, the method comprising providing cells of the mammal with a polypeptide encoded by the nucleic acid according to claim 1 under conditions such that said treatment is effected.
 17. A method according to claim 16 wherein the polypeptide is provided to the cells by expression from encoding nucleic acid administered to the mammal.
 18. An isolated nucleic acid encoding a polypeptide different from the polypeptide of which the amino acid sequence is shown in FIG. 3 (SEQ ID NO:8), wherein the polypeptide: has an actin-binding-domain which has at least 90% amino acid sequence identity with the actin-binding domain of the polypeptide of which the amino acid sequence is shown in FIG. 3 (SEQ ID NO:8), and has a dystrophin-protein-complex-binding-domain which has at least 90% amino acid sequence identity with the dystrophin-protein-complex-binding domain of the polypeptide of which the amino acid sequence is shown in FIG. 3 (SEQ ID NO:8), wherein the nucleotide sequence of the nucleic acid encoding the polypeptide comprises nucleotides 342 to 348 and 1462 to 1486 of FIG. 3 (SEQ ID NO:7), and on expression in a host cell prodces the polypeptide.
 19. The isolated nucleic acid according to claim 18 wherein the amino acid sequence of the polypeptide is that shown in FIG. 9 (SEQ ID NO:10).
 20. The isolated nucleic acid according to claim 19 wherein the nucleotide sequence of the nucleic acid encoding the polypeptide is the coding nucleotide sequence of FIG. 9 (SEQ ID NO:9).
 21. The isolated nucleic acid according to claim 19 wherein the nucleotide sequence of the nucleic acid encoding the polypeptide differs from the coding nucleotide sequence shown in FIG. 9 (SEQ ID NO:9).
 22. A vector comprising the nucleic acid according to claim
 1. 23. The vector according to claim 22 wherein said vector is an expression vector.
 24. A composition comprising the nucleic acid according to claim 18 and a pharmaceutically acceptable excipient.
 25. A cell containing the nucleic acid according to claim 18, which nucleic acid is heterologous to the cell.
 26. A cell according to claim 25 which is a muscle cell.
 27. A cell according to claim 25 wherein said polypeptide is expressed.
 28. A method comprising introducing the nucleic acid according to claim 18 into a cell.
 29. A method according to claim 28 wherein said cell is a muscle cell.
 30. A method according to claim 28 comprising introducing said nucleic acid into said cell in vitro.
 31. A method which includes expressing the coding nucleotide sequence of the nucleic acid according to claim 18 in a cell, which nucleic acid is heterologous to the cell.
 32. The method according to claim 31 wherein said polypeptide is produced, and is then purified or isolated.
 33. The method according to claim 32 wherein the polypeptide is formulated into a composition which comprising a pharmaceutically acceptable excipient, following purification or isolation of the polypeptide.
 34. A method for treating muscular dystrophy in a mammal, the method comprising providing cells of the mammal with a polypeptide encoded by the nucleic acid according to claim 18 under conditions such that said treatment is effected.
 35. A method for treating muscular dystrophy in a mammal, the method comprising providing cells of the mammal with a polypeptide according to claim 19 under conditions such that said treatment is effected. 